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What is IFITM?	interferon-induced transmembrane	[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]	650	568	Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as "-" and "+", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.
How many cysteine residues are contained in the first transmembrane domain of IFITM3?	three	[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]	650	569	Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as "-" and "+", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.
What inhibits S-palmitoylation?	2-bromopalmitic acid (2BP)	[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]	650	570	Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as "-" and "+", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.
What interaction is inhibited by the presence of 2-bromopalmitic acid (2BP)?	IFITM5 with FKBP11	[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]	650	571	Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as "-" and "+", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.
What is a function associated with IFITM5?	bone formation factor.	[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]	650	572	Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as "-" and "+", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.
What regulates the antiviral activity of IFITM3?	S-palmitoylation on the protein	[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]	650	573	Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as "-" and "+", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.
What is another name for IFITM5?	bonerestricted IFITM-like (BRIL) protein	[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]	650	574	Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as "-" and "+", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.
Why is the expression of IFITM5 not promoted by interferons?	the region upstream of the ifitm5 gene lacks the interferon regulatory elements	[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]	650	575	Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as "-" and "+", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.
What is the amino acid similarity between IFITM5 and the other IFITM proteins?	~ 65% similarity	[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]	650	576	Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as "-" and "+", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.
What is the amino acid similarity between IFITM 1, IFITM 2, and IFITM 3?	~ 85% similarity	[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]	650	577	Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as "-" and "+", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.
What amino acid might be involved in calcium binding in the C-terminal region of a protein?	aspartate	[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]	650	580	Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE \| does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as "-" and "+", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.
What is required for a Hepatitis B infection in cells?	both intracellular and cell-surface factors	[ "Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus HBV and its satellite hepatitis D virus HDV still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma HCC cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors.", "Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction.", "The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender . HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s .", "Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al.", "Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor s . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS.", "In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR.", "Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor.", "NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected .", "Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one hundreds or thousands fold viral amplification . For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro.", "For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\".", "The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment." ]	1,552	2,996	Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus HBV and its satellite hepatitis D virus HDV still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma HCC cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender . HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor s . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one hundreds or thousands fold viral amplification . For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment.
What regulates the broad, but less specific, virus-cell interaction in a hepatitis B infection?	heparan sulfates in the membrane proteins	[ "Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus HBV and its satellite hepatitis D virus HDV still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma HCC cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors.", "Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction.", "The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender . HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s .", "Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al.", "Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor s . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS.", "In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR.", "Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor.", "NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected .", "Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one hundreds or thousands fold viral amplification . For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro.", "For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\".", "The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment." ]	1,552	2,997	Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus HBV and its satellite hepatitis D virus HDV still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma HCC cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender . HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor s . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one hundreds or thousands fold viral amplification . For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment.
Which protein domain of the Hepatitis B envelope is necessary for infection?	Nterminus of HBV preS1 (amino acids 1-47)	[ "Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus HBV and its satellite hepatitis D virus HDV still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma HCC cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors.", "Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction.", "The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender . HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s .", "Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al.", "Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor s . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS.", "In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR.", "Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor.", "NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected .", "Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one hundreds or thousands fold viral amplification . For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro.", "For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\".", "The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment." ]	1,552	2,998	Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus HBV and its satellite hepatitis D virus HDV still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma HCC cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender . HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor s . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one hundreds or thousands fold viral amplification . For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment.
Where is NTCP located in the body?	lateral surface (canalicular) of hepatocytes	[ "Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus HBV and its satellite hepatitis D virus HDV still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma HCC cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors.", "Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction.", "The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender . HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s .", "Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al.", "Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor s . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS.", "In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR.", "Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor.", "NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected .", "Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one hundreds or thousands fold viral amplification . For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro.", "For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\".", "The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment." ]	1,552	2,999	Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus HBV and its satellite hepatitis D virus HDV still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma HCC cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender . HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor s . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one hundreds or thousands fold viral amplification . For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment.
What does the NTCP protein mediate?	bile acid transport	[ "Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus HBV and its satellite hepatitis D virus HDV still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma HCC cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors.", "Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction.", "The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender . HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s .", "Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al.", "Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor s . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS.", "In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR.", "Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor.", "NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected .", "Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one hundreds or thousands fold viral amplification . For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro.", "For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\".", "The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment." ]	1,552	3,000	Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus HBV and its satellite hepatitis D virus HDV still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma HCC cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender . HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor s . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one hundreds or thousands fold viral amplification . For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment.
Is NTCP sufficient to allow HBV infection?	not sufficient	[ "Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus HBV and its satellite hepatitis D virus HDV still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma HCC cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors.", "Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction.", "The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender . HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s .", "Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al.", "Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor s . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS.", "In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR.", "Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor.", "NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected .", "Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one hundreds or thousands fold viral amplification . For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro.", "For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\".", "The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment." ]	1,552	3,001	Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus HBV and its satellite hepatitis D virus HDV still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma HCC cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender . HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor s . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one hundreds or thousands fold viral amplification . For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment.
Why is NTCP thought to not be sufficient for HBV infection?	the majority of HepaRG cells were found to express NPCT but not to be infected	[ "Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus HBV and its satellite hepatitis D virus HDV still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma HCC cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors.", "Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction.", "The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender . HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s .", "Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al.", "Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor s . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS.", "In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR.", "Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor.", "NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected .", "Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one hundreds or thousands fold viral amplification . For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro.", "For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\".", "The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment." ]	1,552	3,002	Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus HBV and its satellite hepatitis D virus HDV still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma HCC cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender . HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor s . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one hundreds or thousands fold viral amplification . For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment.
What method is useful in administering small molecules for systemic delivery to the body?	Intranasal	[ "RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.", "Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.", "Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.", "Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.", "It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .", "Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .", "Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.", "Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.", "Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.", "Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.", "Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.", "In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.", "A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.", "For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.", "Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.", "Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.", "The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.", "In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.", "For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.", "The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .", "The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.", "Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.", "The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .", "Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.", ". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .", "To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.", "Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.", "Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .", "An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .", "Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.", "This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .", "Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.", "Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.", "Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.", "Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.", "The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.", "Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.", "Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .", "Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .", "ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.", "Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.", "RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.", "Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .", "PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .", "These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .", "COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.", "Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.", "nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.", "Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.", "The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.", "However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .", "The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .", "It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.", "In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .", "MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.", "This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .", "There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.", "Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.", "Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.", "A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .", "Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.", "Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.", "In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .", "Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .", "Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.", "Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .", "The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.", "MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.", "Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .", "Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.", "It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.", "They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.", "This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.", "Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.", "Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.", "A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.", "Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients." ]	641	1,612	RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural "remodeling" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer "naked siRNAs" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term "naked siRNAs" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or "LNA-antimiRs" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients.
Why is the nasal mucosa useful in the delivery of small molecules into the body?	the surface area can result in rapid absorption of the medication into the blood	[ "RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.", "Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.", "Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.", "Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.", "It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .", "Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .", "Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.", "Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.", "Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.", "Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.", "Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.", "In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.", "A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.", "For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.", "Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.", "Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.", "The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.", "In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.", "For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.", "The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .", "The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.", "Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.", "The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .", "Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.", ". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .", "To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.", "Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.", "Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .", "An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .", "Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.", "This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .", "Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.", "Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.", "Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.", "Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.", "The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.", "Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.", "Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .", "Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .", "ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.", "Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.", "RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.", "Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .", "PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .", "These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .", "COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.", "Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.", "nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.", "Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.", "The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.", "However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .", "The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .", "It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.", "In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .", "MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.", "This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .", "There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.", "Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.", "Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.", "A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .", "Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.", "Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.", "In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .", "Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .", "Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.", "Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .", "The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.", "MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.", "Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .", "Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.", "It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.", "They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.", "This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.", "Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.", "Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.", "A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.", "Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients." ]	641	1,615	RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural "remodeling" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer "naked siRNAs" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term "naked siRNAs" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or "LNA-antimiRs" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients.
What are the most common methods of inhaled delivery of medications?	Metered dose inhalers (MDIs) and dry powder inhalers (DPIs)	[ "RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.", "Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.", "Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.", "Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.", "It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .", "Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .", "Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.", "Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.", "Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.", "Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.", "Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.", "In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.", "A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.", "For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.", "Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.", "Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.", "The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.", "In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.", "For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.", "The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .", "The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.", "Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.", "The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .", "Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.", ". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .", "To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.", "Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.", "Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .", "An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .", "Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.", "This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .", "Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.", "Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.", "Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.", "Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.", "The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.", "Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.", "Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .", "Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .", "ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.", "Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.", "RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.", "Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .", "PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .", "These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .", "COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.", "Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.", "nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.", "Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.", "The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.", "However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .", "The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .", "It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.", "In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .", "MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.", "This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .", "There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.", "Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.", "Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.", "A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .", "Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.", "Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.", "In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .", "Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .", "Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.", "Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .", "The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.", "MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.", "Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .", "Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.", "It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.", "They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.", "This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.", "Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.", "Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.", "A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.", "Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients." ]	641	1,616	RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural "remodeling" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer "naked siRNAs" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term "naked siRNAs" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or "LNA-antimiRs" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients.
What medications have shown good promise to in vivo delivery via dry powder inhalers?	insulin and low-molecular-weight heparin	[ "RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.", "Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.", "Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.", "Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.", "It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .", "Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .", "Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.", "Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.", "Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.", "Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.", "Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.", "In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.", "A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.", "For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.", "Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.", "Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.", "The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.", "In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.", "For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.", "The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .", "The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.", "Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.", "The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .", "Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.", ". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .", "To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.", "Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.", "Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .", "An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .", "Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.", "This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .", "Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.", "Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.", "Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.", "Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.", "The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.", "Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.", "Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .", "Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .", "ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.", "Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.", "RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.", "Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .", "PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .", "These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .", "COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.", "Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.", "nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.", "Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.", "The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.", "However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .", "The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .", "It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.", "In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .", "MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.", "This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .", "There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.", "Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.", "Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.", "A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .", "Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.", "Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.", "In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .", "Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .", "Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.", "Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .", "The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.", "MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.", "Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .", "Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.", "It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.", "They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.", "This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.", "Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.", "Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.", "A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.", "Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients." ]	641	1,617	RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural "remodeling" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer "naked siRNAs" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term "naked siRNAs" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or "LNA-antimiRs" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients.
How are siRNAs typically delivered for systemic effect?	intratracheal or intranasal delivery	[ "RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.", "Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.", "Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.", "Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.", "It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .", "Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .", "Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.", "Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.", "Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.", "Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.", "Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.", "In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.", "A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.", "For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.", "Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.", "Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.", "The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.", "In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.", "For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.", "The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .", "The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.", "Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.", "The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .", "Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.", ". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .", "To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.", "Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.", "Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .", "An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .", "Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.", "This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .", "Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.", "Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.", "Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.", "Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.", "The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.", "Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.", "Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .", "Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .", "ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.", "Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.", "RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.", "Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .", "PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .", "These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .", "COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.", "Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.", "nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.", "Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.", "The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.", "However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .", "The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .", "It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.", "In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .", "MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.", "This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .", "There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.", "Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.", "Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.", "A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .", "Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.", "Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.", "In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .", "Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .", "Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.", "Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .", "The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.", "MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.", "Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .", "Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.", "It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.", "They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.", "This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.", "Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.", "Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.", "A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.", "Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients." ]	641	1,618	RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural "remodeling" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer "naked siRNAs" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term "naked siRNAs" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or "LNA-antimiRs" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients.
What structures form the human airway?	respiratory bronchioles, alveolar ducts, and alveolar sacs	[ "RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.", "Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.", "Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.", "Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.", "It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .", "Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .", "Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.", "Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.", "Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.", "Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.", "Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.", "In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.", "A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.", "For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.", "Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.", "Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.", "The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.", "In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.", "For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.", "The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .", "The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.", "Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.", "The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .", "Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.", ". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .", "To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.", "Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.", "Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .", "An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .", "Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.", "This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .", "Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.", "Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.", "Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.", "Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.", "The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.", "Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.", "Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .", "Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .", "ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.", "Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.", "RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.", "Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .", "PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .", "These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .", "COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.", "Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.", "nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.", "Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.", "The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.", "However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .", "The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .", "It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.", "In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .", "MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.", "This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .", "There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.", "Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.", "Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.", "A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .", "Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.", "Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.", "In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .", "Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .", "Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.", "Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .", "The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.", "MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.", "Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .", "Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.", "It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.", "They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.", "This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.", "Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.", "Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.", "A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.", "Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients." ]	641	1,619	RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural "remodeling" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer "naked siRNAs" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term "naked siRNAs" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or "LNA-antimiRs" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients.
What size of particle has been shown to be most effective in the delivery to the lower airway?	1-5 μm	[ "RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.", "Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.", "Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.", "Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.", "It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .", "Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .", "Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.", "Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.", "Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.", "Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.", "Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.", "In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.", "A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.", "For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.", "Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.", "Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.", "The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.", "In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.", "For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.", "The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .", "The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.", "Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.", "The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .", "Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.", ". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .", "To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.", "Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.", "Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .", "An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .", "Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.", "This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .", "Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.", "Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.", "Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.", "Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.", "The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.", "Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.", "Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .", "Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .", "ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.", "Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.", "RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.", "Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .", "PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .", "These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .", "COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.", "Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.", "nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.", "Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.", "The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.", "However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .", "The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .", "It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.", "In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .", "MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.", "This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .", "There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.", "Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.", "Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.", "A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .", "Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.", "Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.", "In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .", "Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .", "Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.", "Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .", "The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.", "MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.", "Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .", "Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.", "It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.", "They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.", "This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.", "Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.", "Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.", "A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.", "Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients." ]	641	1,620	RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural "remodeling" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer "naked siRNAs" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term "naked siRNAs" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or "LNA-antimiRs" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients.
What are the essential conditions in siRNA delivery to effectively produce gene silencing in the lungs?	delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration	[ "RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.", "Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.", "Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.", "Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.", "It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .", "Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .", "Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.", "Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.", "Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.", "Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.", "Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.", "In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.", "A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.", "For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.", "Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.", "Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.", "The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.", "In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.", "For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.", "The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .", "The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.", "Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.", "The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .", "Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.", ". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .", "To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.", "Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.", "Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .", "An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .", "Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.", "This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .", "Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.", "Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.", "Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.", "Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.", "The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.", "Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.", "Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .", "Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .", "ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.", "Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.", "RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.", "Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .", "PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .", "These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .", "COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.", "Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.", "nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.", "Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.", "The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.", "However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .", "The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .", "It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.", "In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .", "MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.", "This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .", "There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.", "Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.", "Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.", "A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .", "Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.", "Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.", "In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .", "Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .", "Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.", "Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .", "The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.", "MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.", "Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .", "Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.", "It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.", "They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.", "This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.", "Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.", "Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.", "A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.", "Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients." ]	641	1,621	RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural "remodeling" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer "naked siRNAs" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term "naked siRNAs" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or "LNA-antimiRs" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients.
Which type of bacteria are implicated in carrying genes of drug resistance?	Gammaproteobacteria	[ "There have been an increasing number of reports implicating Gammaproteobacteria as often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand-washing sink lab gallery to model dispersion of green fluorescent protein GFP -expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-expressing E. coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas <30 in.", "However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas <30 in. during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient.", "We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient. IMPORTANCE Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug-resistant bacteria, which then results in hospital-acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks.", "Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic-resistant bacteria that can thrive in wastewater environments and cause infections in vulnerable patients. Text: D espite early reports . . . . .", "Text: D espite early reports . . . . . , the premise that hand-wash sink traps can act as reservoirs of bacteria that cause nosocomial infections has been frequently overlooked. There has recently been an alarming increase in sink-related outbreaks worldwide, with many reports establishing an observational link . . . . . . . . . A sink often operates as an open conduit to wastewater in a patient care area that is often in the same room as the patient. Health care establishments often invest in desperate interventions to deal with nosocomial outbreaks. The preferred method for addressing most of the environmentrelated transmission is to employ enhanced cleaning using chemical and physical agents . .", "The preferred method for addressing most of the environmentrelated transmission is to employ enhanced cleaning using chemical and physical agents . . Unfortunately, routine approaches are inefficient in completely eliminating drug-resistant Gammaproteobacteria in an inaccessible microbiologically active area such as a sink trap 6, . . . . . . The wet, humid, and relatively protected environment in a sink trap favors the formation of rich stable microbial communities . . These communities will be exposed to liquids and waste that are discarded in a sink and may include antimicrobials, discarded beverages, soap, presumably pathogenic bacteria from health care workers' hands, and other items.", ". These communities will be exposed to liquids and waste that are discarded in a sink and may include antimicrobials, discarded beverages, soap, presumably pathogenic bacteria from health care workers' hands, and other items. In short, sink traps could serve as a breeding ground for opportunistic and highly antimicrobial-resistant bacteria that cannot be easily cleaned or removed . . . . . . . There are many reports of a genetic association between pathogens found in sink traps and those found in patients . . However, surprisingly little work has been done to understand the microscale transmission dynamics.", ". However, surprisingly little work has been done to understand the microscale transmission dynamics. It was previously demonstrated using a suspension of fluorescent particles Glo Germ; Glo Germ Co., Moab, UT that material injected into the P-trap gets dispersed around a hand-washing sink . . This result however has not been replicated hitherto in the follow-up studies. Dispersion has never been investigated with living organisms.", ". This result however has not been replicated hitherto in the follow-up studies. Dispersion has never been investigated with living organisms. Ultimately, many details remain unaddressed surrounding the spread of Enterobacteriaceae in sink-trap wastewater systems: i can organisms grow retrograde from the P-trap water to the sink strainer, ii can organisms spread from one sink to another along the internal surfaces of pipes with shared drainage systems, and iii which portion of a colonized drain pipe results in dispersion into the sink bowl during a hand-washing event? We aim to better understand the dispersion dynamics of Gammaproteobacteria living in the wastewater of a sink strainer and P-trap into an area where patients and health care workers could be exposed. To study this dynamic, we used a surrogate organism that could be easily tracked while remaining in the Enterobacteriaceae family, where some of the most concerning threats in antimicrobial resistance are developing .", "We aim to better understand the dispersion dynamics of Gammaproteobacteria living in the wastewater of a sink strainer and P-trap into an area where patients and health care workers could be exposed. To study this dynamic, we used a surrogate organism that could be easily tracked while remaining in the Enterobacteriaceae family, where some of the most concerning threats in antimicrobial resistance are developing . . Growth and colonization of GFP-expressing E. coli in the P-trap. In the first 14 days following the installation of the P-trap with established green fluorescent protein GFP -expressing Escherichia coli and just water running from the faucet, GFPexpressing E. coli was not detected in the tailpipe beyond 1.5 in. above the liquid level in the P-trap.", "In the first 14 days following the installation of the P-trap with established green fluorescent protein GFP -expressing Escherichia coli and just water running from the faucet, GFPexpressing E. coli was not detected in the tailpipe beyond 1.5 in. above the liquid level in the P-trap. GFP-expressing E. coli, however, was found to be viable in the P-trap without any nutrients added. A nutrient regimen was then instituted to understand the influence of nutrients on mobility and upward growth. The addition of tryptic soy broth TSB promoted GFP-expressing E. coli growth as early as day 1, with growth observed in the tailpipe 2 in. above the liquid surface in the P-trap Table 1 .", "The addition of tryptic soy broth TSB promoted GFP-expressing E. coli growth as early as day 1, with growth observed in the tailpipe 2 in. above the liquid surface in the P-trap Table 1 . On day 7, the strainer ϳ8 in. above the liquid in the P-trap was found to be colonized with GFP-expressing E. coli. This translates to an average growth rate of 1 in./day along the length of the tailpipe with the addition of nutrients and without faucet operation. GFP-expressing E. coli was not detected in the faucet water. Sink-to-sink transmission of bacteria.", "GFP-expressing E. coli was not detected in the faucet water. Sink-to-sink transmission of bacteria. In these experiments, a flanking sink sink 5 was the only P-trap inoculated with GFP-expressing E. coli and therefore was the sole source for transmission to the connected sinks. Starting with a lower inoculum concentration 10 3 CFU/ml in sink 5, on day 7, GFP-expressing E. coli was detected in the sink 2 and sink 3 P-traps Fig. 1a . With inoculum concentrations of 10 6 CFU/ml and Ͼ10 10 CFU/ml in sink 5, all of the sink P-traps in the sink gallery with the exception of sink 1 were found to be colonized with GFP-expressing E. coli after 7 days Fig.", "1a . With inoculum concentrations of 10 6 CFU/ml and Ͼ10 10 CFU/ml in sink 5, all of the sink P-traps in the sink gallery with the exception of sink 1 were found to be colonized with GFP-expressing E. coli after 7 days Fig. 1b and c . Faucet water and aerators tested negative for GFP-expressing E. coli. Irrespective of the starting inoculum concentration, on day 7 the highest level of colonization was recorded in the sink 3 P-trap. After day 7, when the nutrient regimen described previously was followed for an additional 7 days in each of the sinks in the sink gallery with an inoculum concentration of Ͼ10 10 CFU/ml, GFP-expressing E. coli was detected in the strainers of sinks 2 and 3 on day 14.", "Irrespective of the starting inoculum concentration, on day 7 the highest level of colonization was recorded in the sink 3 P-trap. After day 7, when the nutrient regimen described previously was followed for an additional 7 days in each of the sinks in the sink gallery with an inoculum concentration of Ͼ10 10 CFU/ml, GFP-expressing E. coli was detected in the strainers of sinks 2 and 3 on day 14. This finding validated the upward growth and growth rate in the tailpipe when nutrients were added. Nonfluorescent colonies were occasionally observed in the P-trap water samples collected from the sinks, which were subsequently identified to be Pseudomonas sp. or Stenotrophomonas maltophilia, and fluorescent colonies were confirmed to be E. coli. Dispersion of microspheres from sinks.", "or Stenotrophomonas maltophilia, and fluorescent colonies were confirmed to be E. coli. Dispersion of microspheres from sinks. In the first dispersion experiment, when fluorescent microspheres were inoculated into the offset drain tailpiece only 4 in. below the strainer, no microspheres were detected on the polyester sheets placed on the counter space. However, when the sink bowl was coated with the microspheres, polyester sheets overlaid on the counter space captured the dispersed microspheres caused by the faucet operation. Dispersion was observed on almost all zones of the sink counter space Fig. 2 .", "Dispersion was observed on almost all zones of the sink counter space Fig. 2 . Relatively higher levels of dispersion were observed along the major and minor axes of the elliptical sink bowl zones 2, 5, 6, 9, 11, and 12 . Anterior corners of the sink counter space zones 4 and 7 , which were most distant from the impact of water in the sink bowl, received the lowest dispersion. Dispersion of GFP-expressing E. coli from sinks. Initially the P-trap alone was inoculated with GFP-expressing E. coli and carefully installed, keeping the tailpipe and strainer free of GFP-expressing E. coli before operating the faucets.", "Dispersion of GFP-expressing E. coli from sinks. Initially the P-trap alone was inoculated with GFP-expressing E. coli and carefully installed, keeping the tailpipe and strainer free of GFP-expressing E. coli before operating the faucets. No fluorescent CFU were observed on the plates placed on the counter or attached to the bowl surface after faucet operation. Similarly, no fluorescent CFU were detected when GFPexpressing E. coli was inoculated into the offset drain tailpiece only 4 in. below the strainer. Interestingly, when there was conspicuous water backup over the strainer as a result of a higher water flow rate from the faucet than the drainage rate from the P-trap, dispersal was detected on the plates attached to the bowl surface.", "below the strainer. Interestingly, when there was conspicuous water backup over the strainer as a result of a higher water flow rate from the faucet than the drainage rate from the P-trap, dispersal was detected on the plates attached to the bowl surface. The dispersion pattern recorded when the sink bowl was coated with GFPexpressing E. coli was comparable to the pattern recorded when the sink bowl was coated with fluorescent microspheres Fig. 2 . Dispersion was significantly higher along the axes zones 6, 9, 11, and 12 and lower at the corners of the sink counter space zones 4, 7, and 10 . In contrast, dispersion of GFP-expressing E. coli caused by the faucet operation was much more extensive when the strainer was allowed to be colonized with GFPexpressing E. coli prior to the dispersion experiment.", "Dispersion was significantly higher along the axes zones 6, 9, 11, and 12 and lower at the corners of the sink counter space zones 4, 7, and 10 . In contrast, dispersion of GFP-expressing E. coli caused by the faucet operation was much more extensive when the strainer was allowed to be colonized with GFPexpressing E. coli prior to the dispersion experiment. In addition to the sink counter space, we measured dispersion to the sink bowl, faucet, faucet handles, splatter shields, and the extended counter surface. Dispersion of GFP-expressing E. coli was highest on the plates attached to the sink bowl Fig. 3b . Further, dispersion was greater along the Ϫ\" and \"ϩ\" denote the absence and presence of GFP-expressing E. coli, respectively.", "3b . Further, dispersion was greater along the Ϫ\" and \"ϩ\" denote the absence and presence of GFP-expressing E. coli, respectively. minor axis of the sink bowl Fig. 3b , zones B3, B4, and B10 than along the major axis of the sink bowl, associated with a shorter distance from the strike point of the faucet water to the bowl along this axis. The next highest CFU count from the dispersal was recorded on the counter area near the faucets Fig. 3a , zones 12 and 11 . A similar pattern of higher dispersion near the faucets and lower dispersion at the corners of the counter space Fig.", "3a , zones 12 and 11 . A similar pattern of higher dispersion near the faucets and lower dispersion at the corners of the counter space Fig. 3a , zones 4, 7, and 10 was also observed using microspheres. Dispersion was also recorded in other zones of the counter space, on the Plexiglas splatter shields, faucets, faucet handles, and extended surface Fig. 3c . There were no GFP-expressing E. coli CFU recorded on plates placed beyond 30 in. from the strainer, demarcating the range of dispersion under these experimental conditions. Table 2 gives a summary of the total distribution loads recorded using fluorescent microspheres and GFP-expressing E. coli across each experiment.", "from the strainer, demarcating the range of dispersion under these experimental conditions. Table 2 gives a summary of the total distribution loads recorded using fluorescent microspheres and GFP-expressing E. coli across each experiment. The loads of dispersion on the sink counter were comparable when the sink bowl was coated with microspheres or GFP-expressing E. coli before the faucet operation. Although the dispersion load on the sink counter was lower when the sink strainer was colonized, it is interesting to note that the sink bowl received the highest dispersion. To mimic dispersion in a hospital setting, we first investigated whether GFPexpressing E. coli would establish consistent colonization in a sink trap as many other Gammaproteobacteria implicated in nosocomial outbreaks have done . .", "To mimic dispersion in a hospital setting, we first investigated whether GFPexpressing E. coli would establish consistent colonization in a sink trap as many other Gammaproteobacteria implicated in nosocomial outbreaks have done . . Many recent reports demonstrate that P-traps become colonized with highly consequential Gammaproteobacteria, which then results in nosocomial transmission . . The retained water in a sink P-trap is present to provide a water barrier to prevent off-gassing of sewer smell, but it may inadvertently provide favorable conditions for pathogenic and opportunistic antibiotic-resistant microorganisms to survive and develop resilient biofilms . . However, the mechanism of dispersal of the bacteria in the P-trap to patients or the surrounding health care area had not been fully elucidated.", ". However, the mechanism of dispersal of the bacteria in the P-trap to patients or the surrounding health care area had not been fully elucidated. We began with the hypothesis that the bacteria originate from the P-trap via droplet creation when the water from the faucet hits the P-trap water, thus contaminating the sink bowl and the surrounding area. The finding supporting this theory had been previously reported using Glo Germ particles . . However, in the present study with careful attention to avoid strainer and tail piece contamination, the dispersal directly from the sink P-trap with either microspheres or GFP-expressing E. coli could not be reproduced as previously reported . .", "However, in the present study with careful attention to avoid strainer and tail piece contamination, the dispersal directly from the sink P-trap with either microspheres or GFP-expressing E. coli could not be reproduced as previously reported . . Rather this work demonstrates a different, more staged mode of transmission from a P-trap reservoir to the sink and surrounding environment. GFP-expressing E. coli in the P-trap alone was sustained for 14 days but did not grow or mobilize up the tailpipe to the strainer with just intermittent water exposure. However, when nutrients were subsequently added to the system, the organisms rapidly grew up the tailpipe to the strainer at approximately an inch per day. In a real-world setting, motility of bacteria inside the tailpipe is restricted to relatively sporadic and brief wetting events in which swimming is an opportunity to colonize new surfaces.", "However, when nutrients were subsequently added to the system, the organisms rapidly grew up the tailpipe to the strainer at approximately an inch per day. In a real-world setting, motility of bacteria inside the tailpipe is restricted to relatively sporadic and brief wetting events in which swimming is an opportunity to colonize new surfaces. It is assumed that once established, the biofilm promotes the upward growth of GFP-expressing E. coli in the tailpipe at an accelerated rate. The nutrient regimen that promoted colonization in our model reflects our and others' observations of items commonly disposed of in hospital sinks intravenous fluids, feeding supplements, and leftover beverages . . Transmission of bacteria between sinks via a common pipe was a key finding in this study as this highlights the concept that premise plumbing may be a more continuous system with shared microbiology than a single isolated sink.", ". Transmission of bacteria between sinks via a common pipe was a key finding in this study as this highlights the concept that premise plumbing may be a more continuous system with shared microbiology than a single isolated sink. The sink gallery used in this study provided a unique in situ advantage to investigate sink-to-sink transmission of bacteria through common drains. The two possible mechanisms for P-trap strainers becoming colonized are seeding of organisms from above and retrograde spread of organisms along common pipes in a hospital wastewater infrastructure. Here we demonstrate that it is possible for GFP-expressing E. coli to contaminate adjacent P-traps with just time and water given a standard U.S. code piping rise of 0.25 ft/ft. Sink-to-sink or retrograde transmission may explain the recurrence of pathogen colonization following intervention strategies like disinfection or replacement of plumbing .", "Here we demonstrate that it is possible for GFP-expressing E. coli to contaminate adjacent P-traps with just time and water given a standard U.S. code piping rise of 0.25 ft/ft. Sink-to-sink or retrograde transmission may explain the recurrence of pathogen colonization following intervention strategies like disinfection or replacement of plumbing . . Sink 3 was lowest on the slope in the drain line see Fig. 4 with arguably the most opportunity for reflux and retrograde wetting. Sink 1, on the other hand, was farthest away from the source sink 5 , and its P-trap had the greatest incline in the drain line connecting the sinks, which could perhaps contribute to the reasons there was no GFP-expressing E. coli colonization detected in it after 7 days.", "4 with arguably the most opportunity for reflux and retrograde wetting. Sink 1, on the other hand, was farthest away from the source sink 5 , and its P-trap had the greatest incline in the drain line connecting the sinks, which could perhaps contribute to the reasons there was no GFP-expressing E. coli colonization detected in it after 7 days. There has been more investigation about microbiologic dynamics of infectious viral particles such as those of severe acute respiratory syndrome SARS and Ebola viruses through premise plumbing systems . . . . However, the microbiology, sustainability, and dynamics might be very different, although the backflow and inoculation issues could have some parallels when comparing viruses to bacteria.", ". . However, the microbiology, sustainability, and dynamics might be very different, although the backflow and inoculation issues could have some parallels when comparing viruses to bacteria. As Enterobacteriaceae can either multiply or remain viable for long periods of time in biofilms coating the interior of P-traps and the connected plumbing, it may not be sustainable to target any intervention limited to a single isolated sink as a source of a particular pathogen. Data from different dispersal experiments suggest that although P-traps can act as the source or the reservoir of pathogens, the physical presence of the organism in the sink bowl or colonization of the strainer is necessary for the dispersal to occur. Colonization of strainers or drains reported in earlier studies .", "Data from different dispersal experiments suggest that although P-traps can act as the source or the reservoir of pathogens, the physical presence of the organism in the sink bowl or colonization of the strainer is necessary for the dispersal to occur. Colonization of strainers or drains reported in earlier studies . was perhaps a result of ascending biofilm growth from the P-trap to the strainer or introduction through contaminated fluids. Many of the studies used swab samples, which likely sampled the strainer rather than P-trap water . . Once the strainer was colonized, the water from the faucet resulted in GFP-expressing E. coli dispersion in the bowl and to the surrounding surfaces of up to 30 in.", ". Once the strainer was colonized, the water from the faucet resulted in GFP-expressing E. coli dispersion in the bowl and to the surrounding surfaces of up to 30 in. The range of dispersal . . Greater dispersal near the faucet may be attributed to the specific designs of the sink bowl and faucet in this study, which determine the contact angle of water impact. This is an important finding since many sinks in hospitals are similar in design, with faucet handles representing a high-touch surface for the sink users . . It can also be concluded from the dispersion experiments that secondary and successive dispersals would likely increase the degree and the scope of dispersion.", ". It can also be concluded from the dispersion experiments that secondary and successive dispersals would likely increase the degree and the scope of dispersion. There are several limitations to this work. First the use of similar sink bowls across these sinks only examines the dispersion pattern of this particular sink design. Similarly the sink-to-sink transmission may not be applicable to all wastewater plumbing systems as the fixtures on the pipe are very close together, unlike most layouts in health care settings. However, we speculate that transmission could occur on larger systems over greater time scales, especially if heavy nutrient and contamination loads were also included.", "Similarly the sink-to-sink transmission may not be applicable to all wastewater plumbing systems as the fixtures on the pipe are very close together, unlike most layouts in health care settings. However, we speculate that transmission could occur on larger systems over greater time scales, especially if heavy nutrient and contamination loads were also included. GFP-expressing E. coli is a laboratory surrogate, and the putative biofilms established in the short time frame of our experiments are unlikely to be as complex or stable as biofilms developed in a hospital wastewater system over many years. However, to address the monomicrobial dominance of the GFP-expressing E. coli added to the system, we kept the system open, and other environmental organisms were able to cocolonize in an attempt to mimic the hospital system. Another limitation was the need to add nutrients to the drain to ensure rapid and robust colonization. We are not clear how widespread the practice of disposing of dextrose-containing intravenous fluids or leftover beverages in the hand-wash sinks is; however, we have observed this practice, and anecdotally it appears to be relatively common in the United States.", "Another limitation was the need to add nutrients to the drain to ensure rapid and robust colonization. We are not clear how widespread the practice of disposing of dextrose-containing intravenous fluids or leftover beverages in the hand-wash sinks is; however, we have observed this practice, and anecdotally it appears to be relatively common in the United States. We also did not completely characterize the droplet sizes, nor do we demonstrate air sampling to understand if the dispersion is only droplet or if there are also aerosols that contain GFP-expressing E. coli. This would require additional testing and is planned as future work. In summary, this work for the first time better models the mechanisms of spread of multidrug-resistant pathogens arising from the sink drain and infecting patients. Droplet dispersion from the P-trap does not happen directly.", "In summary, this work for the first time better models the mechanisms of spread of multidrug-resistant pathogens arising from the sink drain and infecting patients. Droplet dispersion from the P-trap does not happen directly. Rather it is a multistage process: dispersal originates from the strainer and/or the bowl after growth of the biofilm up from the microbial reservoir of the P-trap. We also demonstrate sink-to-sink transmission via a common sanitary pipe. This work could have implications for patient safety, infection control, and interventions as well as the design of future hospital plumbing systems to eliminate this mode of transmission to vulnerable hospitalized patients. Sink gallery design.", "This work could have implications for patient safety, infection control, and interventions as well as the design of future hospital plumbing systems to eliminate this mode of transmission to vulnerable hospitalized patients. Sink gallery design. A dedicated sink gallery was set up to simulate hospital hand-washing sinks. The gallery was comprised of five sink modules assembled next to each other Fig. 4 . The five hand-wash sink stations were identical in bowl designs and dimensions and were modeled from the most common intensive care unit hand-washing sink type in the acute care hospital at the University of Virginia Medical Center. Partitions made of 24-in.-high Plexiglas sheet were installed between the sinks to prevent splatter and cross contamination.", "The five hand-wash sink stations were identical in bowl designs and dimensions and were modeled from the most common intensive care unit hand-washing sink type in the acute care hospital at the University of Virginia Medical Center. Partitions made of 24-in.-high Plexiglas sheet were installed between the sinks to prevent splatter and cross contamination. Each sink module was built with Corian integrated sink/countertops without an overflow and fitted with an 8-in. centerset 2-handle gooseneck faucet Elkay, Oak Brook, IL . The drain line Dearborn Brass-Oatey, Cleveland, OH . All of the fixtures were made of brass with chrome plating. Each of the sink P-traps was connected to a 3-in.", "All of the fixtures were made of brass with chrome plating. Each of the sink P-traps was connected to a 3-in. common cast-iron pipe sloping into a T-joint leading into the building sanitary line located behind sink 3 Fig. 4 . Inoculation, growth, and establishment of GFP-expressing E. coli in sink P-traps. For the GFP-expressing E. coli strain ATCC 25922GFP , the green fluorescent protein GFP gene is contained on a plasmid that also contains an ampicillin resistance gene. A single isolated colony of GFP-expressing E. coli grown from a Ϫ80°C stock was inoculated into 5 ml tryptic soy broth TSB Becton, Dickinson and Company, Sparks, MD containing 100 g/ml ampicillin ATCC medium 2855 .", "For the GFP-expressing E. coli strain ATCC 25922GFP , the green fluorescent protein GFP gene is contained on a plasmid that also contains an ampicillin resistance gene. A single isolated colony of GFP-expressing E. coli grown from a Ϫ80°C stock was inoculated into 5 ml tryptic soy broth TSB Becton, Dickinson and Company, Sparks, MD containing 100 g/ml ampicillin ATCC medium 2855 . The inoculum concentration and method varied for each experiment. For establishment of GFP-expressing E. coli in sink P-traps, new autoclaved P-traps were filled with 100 ml 0.1ϫ TSB and inoculated with ϳ10 3 CFU/ml GFPexpressing E. coli. Following inoculation, both ends of the P-traps were covered with perforated Parafilm Bemis, Inc., Oshkosh, WI and allowed to incubate at room temperature 22 Ϯ 2°C for 14 days to facilitate adherent bacterial growth. The medium in the P-trap was decanted and replaced with fresh 0.1ϫ TSB every 48 h. An aliquot of decanted medium and a swab sample from the inner surface of the P-trap were plated on tryptic soy agar Becton, Dickinson and Company, Sparks, MD plates containing 100 g/ml ampicillin TSA to monitor the growth of GFP-expressing E. coli in the P-traps.", "Following inoculation, both ends of the P-traps were covered with perforated Parafilm Bemis, Inc., Oshkosh, WI and allowed to incubate at room temperature 22 Ϯ 2°C for 14 days to facilitate adherent bacterial growth. The medium in the P-trap was decanted and replaced with fresh 0.1ϫ TSB every 48 h. An aliquot of decanted medium and a swab sample from the inner surface of the P-trap were plated on tryptic soy agar Becton, Dickinson and Company, Sparks, MD plates containing 100 g/ml ampicillin TSA to monitor the growth of GFP-expressing E. coli in the P-traps. TSA plates were incubated overnight at 37°C, and CFU fluorescing under UV light were enumerated. All preparatory culturing of GFP-expressing E. coli took place in a separate room from the sink gallery to avoid unintentional contamination. Installation of P-traps colonized with GFP-expressing E.coli. After the 14-day incubation, P-traps were fastened into the plumbing of the sinks Fig.", "Installation of P-traps colonized with GFP-expressing E.coli. After the 14-day incubation, P-traps were fastened into the plumbing of the sinks Fig. 5a . The remainder of the drain line was either autoclaved strainer, tailpipe, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI , maintaining at least 1 min of contact time. After the P-trap was installed, a daily regimen was followed in which 25 ml of TSB followed by 25 ml of 0.9% NaCl solution saline were added in the ratio 1:3 via the strainer Fig. 5b to mimic the potential nutrient exposure in the hospital.", "After the P-trap was installed, a daily regimen was followed in which 25 ml of TSB followed by 25 ml of 0.9% NaCl solution saline were added in the ratio 1:3 via the strainer Fig. 5b to mimic the potential nutrient exposure in the hospital. Sampling and enumeration of GFP-expressing E. coli. To monitor the growth of GFP-expressing E. coli in the plumbing, sampling ports were drilled along the length of the tailpiece between the P-trap and the strainer and the trap arm between the P-trap and the common line . These holes were fitted with size 00 silicone stoppers Cole-Parmer, Vernon Hills, IL Fig. 5a .", "These holes were fitted with size 00 silicone stoppers Cole-Parmer, Vernon Hills, IL Fig. 5a . Sterile cotton swabs Covidien, Mansfield, MA presoaked in saline were inserted through these sampling ports, and samples were collected by turning the swab in a circular motion on the inner surface ϳ20 cm 2 of the tailpipes. Sample swabs were pulse-vortexed in 3 ml saline, and serial dilutions were plated on TSA. The strainer, faucet aerator, and bowl surface were sampled with presoaked swabs and processed as described earlier. Sink-to-sink transmission of bacteria. To investigate sink-to-sink transmission of bacteria, a distal sink sink 5 Fig.", "Sink-to-sink transmission of bacteria. To investigate sink-to-sink transmission of bacteria, a distal sink sink 5 Fig. 4 was fitted with a P-trap inoculated with GFP-expressing E. coli. The effects of different inoculum concentrations of GFP-expressing E. coli-10 3 , 10 6 , and Ͼ10 10 CFU/ml colonized for 14 days -were investigated. Identification to the species level of fluorescent and nonfluorescent colonies identified from mixed pipe cultures was performed using a matrix-assisted laser desorption-ionization MALDI -time of flight MALDI-TOF mass spectrometer Vitek-MS; bioMérieux, Durham, NC . The wastewater paths of sinks 1 to 4 were either autoclaved strainer, tailpipe, P-traps, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI .", "Identification to the species level of fluorescent and nonfluorescent colonies identified from mixed pipe cultures was performed using a matrix-assisted laser desorption-ionization MALDI -time of flight MALDI-TOF mass spectrometer Vitek-MS; bioMérieux, Durham, NC . The wastewater paths of sinks 1 to 4 were either autoclaved strainer, tailpipe, P-traps, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI . Faucets on each of the five sinks were turned on simultaneously for 1 min, supplying water at a flow rate of 8 liters/min, once every 24 h for 7 days. No additional feed to any of the sinks was added during this period of 7 days. On days 0 and 7, P-traps on each of the five sinks were unfastened, and swab samples from the P-trap were collected and processed as described earlier. Dispersion measured using fluorescent microspheres.", "On days 0 and 7, P-traps on each of the five sinks were unfastened, and swab samples from the P-trap were collected and processed as described earlier. Dispersion measured using fluorescent microspheres. Fluoresbrite YO carboxylate microspheres Polysciences, Inc. which had a 1-m diameter and maximum excitation and emission of 529 nm and 546 nm, respectively, were used as a tracer in the preliminary experiments to understand droplet dispersion from the hand-wash sinks. To test whether microspheres could be dispersed from below the sink strainer, a 1-ml suspension of microspheres ϳ10 10 particles was injected through a strainer attached to a Hert 4.5-in. offset drain tailpiece typically used for wheelchair-accessible sinks American Standard, model 7723018.002 Fig. 5c .", "offset drain tailpiece typically used for wheelchair-accessible sinks American Standard, model 7723018.002 Fig. 5c . The vertical distance between the strainer and microsphere suspension injected into the tailpipe was ϳ4 in. Counter space around the sink bowl was thoroughly wiped with alcohol wipes Covidien Webcol 6818; Kendall , and polyester sheets precut to appropriate shapes were placed on the counter to cover the entire sink counter and labeled according to position Fig. 6a . The faucet was turned on for 5 min at a water flow rate of 1.8 to 3.0 liters/min.", "6a . The faucet was turned on for 5 min at a water flow rate of 1.8 to 3.0 liters/min. Polyester sheets were harvested and immediately analyzed using a ChemiDoc MP system Bio-Rad Laboratories, Inc. with an exposure time of 5 s. Fluorescent microspheres were enumerated from the digital micrographs using the Image Lab Software Bio-Rad Laboratories, Inc. . To test whether microspheres could be dispersed from the surface of the sink bowl, the sink bowl was evenly coated with a 20-ml microsphere suspension ϳ10 10 particles/ml using a disposable swab Sage Products, Inc., Cary, IL , and the dispersion experiment was repeated following the protocol described above. To ascertain there was no nonspecific background fluorescence in the sink and/or the water from the faucet, a control using the same protocol but without the fluorescent microspheres was performed before each experiment. Dispersion measured using GFP-expressing E.coli.", "To ascertain there was no nonspecific background fluorescence in the sink and/or the water from the faucet, a control using the same protocol but without the fluorescent microspheres was performed before each experiment. Dispersion measured using GFP-expressing E.coli. Dispersion using GFP-expressing E. coli was investigated in three experiments. To test whether live organisms in the P-trap could be dispersed by running water, ϳ10 10 CFU/ml GFP-expressing E. coli in saline was added to an autoclaved P-trap and fitted into the drain line that was preautoclaved strainer, tailpipe, and trap arms . Similarly, to test whether live organisms could be dispersed from the tailpieces of wheelchair-accessible sinks, a suspension of ϳ10 10 CFU/ml GFP-expressing E. coli was added with a syringe through the strainer into the Hert 4.5-ft offset drain tailpiece Fig. 5c .", "Similarly, to test whether live organisms could be dispersed from the tailpieces of wheelchair-accessible sinks, a suspension of ϳ10 10 CFU/ml GFP-expressing E. coli was added with a syringe through the strainer into the Hert 4.5-ft offset drain tailpiece Fig. 5c . Just as in the microsphere dispersion experiment, the vertical distance between the strainer and GFP-expressing E. coli suspension injected into the tailpipe was ϳ4 in. We next tested whether live organisms from the surface of the sink bowl could be dispersed by running water. The sink bowl surface was evenly coated with an approximately 20-ml suspension of 10 10 CFU/ml GFP-expressing E. coli. Finally, to mimic all of these conditions, a P-trap colonized with GFP-expressing E. coli for 14 days was installed, and a nutrient regimen Fig.", "The sink bowl surface was evenly coated with an approximately 20-ml suspension of 10 10 CFU/ml GFP-expressing E. coli. Finally, to mimic all of these conditions, a P-trap colonized with GFP-expressing E. coli for 14 days was installed, and a nutrient regimen Fig. 5b was followed for 14 days to intentionally promote the GFP-expressing E. coli colonization in the attached tailpipe and strainer. On day 15, the dispersion experiment was performed. Before each of the GFP-expressing E. coli dispersion experiments, the counter space was thoroughly disinfected with Caviwipes-1. TSA plates were then positioned on the sink counter surrounding the bowl and an extension platform Fig. 6b .", "TSA plates were then positioned on the sink counter surrounding the bowl and an extension platform Fig. 6b . Additional plates were attached to the sink bowl, faucets, Plexiglas partitions, and faucet handles using adhesive tape. TSA plates were also placed 3 m away from the sink as negative controls. The faucet was turned on for 5 min with a water flow rate of 1.8 to 3.0 liters/min. Lids of the TSA plates were removed only during faucet operation. Swab samples from the faucet aerators before and after operation were collected and plated on TSA.", "Lids of the TSA plates were removed only during faucet operation. Swab samples from the faucet aerators before and after operation were collected and plated on TSA. Prior to each dispersion experiment, 50 ml water from the faucet was also collected, and aliquots were plated to assess for the presence of GFP-expressing E. coli in source water and ensure cross contamination of GFP-expressing E. coli had not occurred. A control dispersion experiment was also performed using the same protocol prior to GFP-expressing E. coli inoculation in each case. Dispersion per defined area CFU per square centimeter was deduced by dividing the CFU counts in the TSA plate by the surface area of the TSA plate." ]	2,585	544	There have been an increasing number of reports implicating Gammaproteobacteria as often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand-washing sink lab gallery to model dispersion of green fluorescent protein GFP -expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-expressing E. coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas <30 in. during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient. IMPORTANCE Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug-resistant bacteria, which then results in hospital-acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic-resistant bacteria that can thrive in wastewater environments and cause infections in vulnerable patients. Text: D espite early reports . . . . . , the premise that hand-wash sink traps can act as reservoirs of bacteria that cause nosocomial infections has been frequently overlooked. There has recently been an alarming increase in sink-related outbreaks worldwide, with many reports establishing an observational link . . . . . . . . . A sink often operates as an open conduit to wastewater in a patient care area that is often in the same room as the patient. Health care establishments often invest in desperate interventions to deal with nosocomial outbreaks. The preferred method for addressing most of the environmentrelated transmission is to employ enhanced cleaning using chemical and physical agents . . Unfortunately, routine approaches are inefficient in completely eliminating drug-resistant Gammaproteobacteria in an inaccessible microbiologically active area such as a sink trap 6, . . . . . . The wet, humid, and relatively protected environment in a sink trap favors the formation of rich stable microbial communities . . These communities will be exposed to liquids and waste that are discarded in a sink and may include antimicrobials, discarded beverages, soap, presumably pathogenic bacteria from health care workers' hands, and other items. In short, sink traps could serve as a breeding ground for opportunistic and highly antimicrobial-resistant bacteria that cannot be easily cleaned or removed . . . . . . . There are many reports of a genetic association between pathogens found in sink traps and those found in patients . . However, surprisingly little work has been done to understand the microscale transmission dynamics. It was previously demonstrated using a suspension of fluorescent particles Glo Germ; Glo Germ Co., Moab, UT that material injected into the P-trap gets dispersed around a hand-washing sink . . This result however has not been replicated hitherto in the follow-up studies. Dispersion has never been investigated with living organisms. Ultimately, many details remain unaddressed surrounding the spread of Enterobacteriaceae in sink-trap wastewater systems: i can organisms grow retrograde from the P-trap water to the sink strainer, ii can organisms spread from one sink to another along the internal surfaces of pipes with shared drainage systems, and iii which portion of a colonized drain pipe results in dispersion into the sink bowl during a hand-washing event? We aim to better understand the dispersion dynamics of Gammaproteobacteria living in the wastewater of a sink strainer and P-trap into an area where patients and health care workers could be exposed. To study this dynamic, we used a surrogate organism that could be easily tracked while remaining in the Enterobacteriaceae family, where some of the most concerning threats in antimicrobial resistance are developing . . Growth and colonization of GFP-expressing E. coli in the P-trap. In the first 14 days following the installation of the P-trap with established green fluorescent protein GFP -expressing Escherichia coli and just water running from the faucet, GFPexpressing E. coli was not detected in the tailpipe beyond 1.5 in. above the liquid level in the P-trap. GFP-expressing E. coli, however, was found to be viable in the P-trap without any nutrients added. A nutrient regimen was then instituted to understand the influence of nutrients on mobility and upward growth. The addition of tryptic soy broth TSB promoted GFP-expressing E. coli growth as early as day 1, with growth observed in the tailpipe 2 in. above the liquid surface in the P-trap Table 1 . On day 7, the strainer ϳ8 in. above the liquid in the P-trap was found to be colonized with GFP-expressing E. coli. This translates to an average growth rate of 1 in./day along the length of the tailpipe with the addition of nutrients and without faucet operation. GFP-expressing E. coli was not detected in the faucet water. Sink-to-sink transmission of bacteria. In these experiments, a flanking sink sink 5 was the only P-trap inoculated with GFP-expressing E. coli and therefore was the sole source for transmission to the connected sinks. Starting with a lower inoculum concentration 10 3 CFU/ml in sink 5, on day 7, GFP-expressing E. coli was detected in the sink 2 and sink 3 P-traps Fig. 1a . With inoculum concentrations of 10 6 CFU/ml and Ͼ10 10 CFU/ml in sink 5, all of the sink P-traps in the sink gallery with the exception of sink 1 were found to be colonized with GFP-expressing E. coli after 7 days Fig. 1b and c . Faucet water and aerators tested negative for GFP-expressing E. coli. Irrespective of the starting inoculum concentration, on day 7 the highest level of colonization was recorded in the sink 3 P-trap. After day 7, when the nutrient regimen described previously was followed for an additional 7 days in each of the sinks in the sink gallery with an inoculum concentration of Ͼ10 10 CFU/ml, GFP-expressing E. coli was detected in the strainers of sinks 2 and 3 on day 14. This finding validated the upward growth and growth rate in the tailpipe when nutrients were added. Nonfluorescent colonies were occasionally observed in the P-trap water samples collected from the sinks, which were subsequently identified to be Pseudomonas sp. or Stenotrophomonas maltophilia, and fluorescent colonies were confirmed to be E. coli. Dispersion of microspheres from sinks. In the first dispersion experiment, when fluorescent microspheres were inoculated into the offset drain tailpiece only 4 in. below the strainer, no microspheres were detected on the polyester sheets placed on the counter space. However, when the sink bowl was coated with the microspheres, polyester sheets overlaid on the counter space captured the dispersed microspheres caused by the faucet operation. Dispersion was observed on almost all zones of the sink counter space Fig. 2 . Relatively higher levels of dispersion were observed along the major and minor axes of the elliptical sink bowl zones 2, 5, 6, 9, 11, and 12 . Anterior corners of the sink counter space zones 4 and 7 , which were most distant from the impact of water in the sink bowl, received the lowest dispersion. Dispersion of GFP-expressing E. coli from sinks. Initially the P-trap alone was inoculated with GFP-expressing E. coli and carefully installed, keeping the tailpipe and strainer free of GFP-expressing E. coli before operating the faucets. No fluorescent CFU were observed on the plates placed on the counter or attached to the bowl surface after faucet operation. Similarly, no fluorescent CFU were detected when GFPexpressing E. coli was inoculated into the offset drain tailpiece only 4 in. below the strainer. Interestingly, when there was conspicuous water backup over the strainer as a result of a higher water flow rate from the faucet than the drainage rate from the P-trap, dispersal was detected on the plates attached to the bowl surface. The dispersion pattern recorded when the sink bowl was coated with GFPexpressing E. coli was comparable to the pattern recorded when the sink bowl was coated with fluorescent microspheres Fig. 2 . Dispersion was significantly higher along the axes zones 6, 9, 11, and 12 and lower at the corners of the sink counter space zones 4, 7, and 10 . In contrast, dispersion of GFP-expressing E. coli caused by the faucet operation was much more extensive when the strainer was allowed to be colonized with GFPexpressing E. coli prior to the dispersion experiment. In addition to the sink counter space, we measured dispersion to the sink bowl, faucet, faucet handles, splatter shields, and the extended counter surface. Dispersion of GFP-expressing E. coli was highest on the plates attached to the sink bowl Fig. 3b . Further, dispersion was greater along the Ϫ" and "ϩ" denote the absence and presence of GFP-expressing E. coli, respectively. minor axis of the sink bowl Fig. 3b , zones B3, B4, and B10 than along the major axis of the sink bowl, associated with a shorter distance from the strike point of the faucet water to the bowl along this axis. The next highest CFU count from the dispersal was recorded on the counter area near the faucets Fig. 3a , zones 12 and 11 . A similar pattern of higher dispersion near the faucets and lower dispersion at the corners of the counter space Fig. 3a , zones 4, 7, and 10 was also observed using microspheres. Dispersion was also recorded in other zones of the counter space, on the Plexiglas splatter shields, faucets, faucet handles, and extended surface Fig. 3c . There were no GFP-expressing E. coli CFU recorded on plates placed beyond 30 in. from the strainer, demarcating the range of dispersion under these experimental conditions. Table 2 gives a summary of the total distribution loads recorded using fluorescent microspheres and GFP-expressing E. coli across each experiment. The loads of dispersion on the sink counter were comparable when the sink bowl was coated with microspheres or GFP-expressing E. coli before the faucet operation. Although the dispersion load on the sink counter was lower when the sink strainer was colonized, it is interesting to note that the sink bowl received the highest dispersion. To mimic dispersion in a hospital setting, we first investigated whether GFPexpressing E. coli would establish consistent colonization in a sink trap as many other Gammaproteobacteria implicated in nosocomial outbreaks have done . . Many recent reports demonstrate that P-traps become colonized with highly consequential Gammaproteobacteria, which then results in nosocomial transmission . . The retained water in a sink P-trap is present to provide a water barrier to prevent off-gassing of sewer smell, but it may inadvertently provide favorable conditions for pathogenic and opportunistic antibiotic-resistant microorganisms to survive and develop resilient biofilms . . However, the mechanism of dispersal of the bacteria in the P-trap to patients or the surrounding health care area had not been fully elucidated. We began with the hypothesis that the bacteria originate from the P-trap via droplet creation when the water from the faucet hits the P-trap water, thus contaminating the sink bowl and the surrounding area. The finding supporting this theory had been previously reported using Glo Germ particles . . However, in the present study with careful attention to avoid strainer and tail piece contamination, the dispersal directly from the sink P-trap with either microspheres or GFP-expressing E. coli could not be reproduced as previously reported . . Rather this work demonstrates a different, more staged mode of transmission from a P-trap reservoir to the sink and surrounding environment. GFP-expressing E. coli in the P-trap alone was sustained for 14 days but did not grow or mobilize up the tailpipe to the strainer with just intermittent water exposure. However, when nutrients were subsequently added to the system, the organisms rapidly grew up the tailpipe to the strainer at approximately an inch per day. In a real-world setting, motility of bacteria inside the tailpipe is restricted to relatively sporadic and brief wetting events in which swimming is an opportunity to colonize new surfaces. It is assumed that once established, the biofilm promotes the upward growth of GFP-expressing E. coli in the tailpipe at an accelerated rate. The nutrient regimen that promoted colonization in our model reflects our and others' observations of items commonly disposed of in hospital sinks intravenous fluids, feeding supplements, and leftover beverages . . Transmission of bacteria between sinks via a common pipe was a key finding in this study as this highlights the concept that premise plumbing may be a more continuous system with shared microbiology than a single isolated sink. The sink gallery used in this study provided a unique in situ advantage to investigate sink-to-sink transmission of bacteria through common drains. The two possible mechanisms for P-trap strainers becoming colonized are seeding of organisms from above and retrograde spread of organisms along common pipes in a hospital wastewater infrastructure. Here we demonstrate that it is possible for GFP-expressing E. coli to contaminate adjacent P-traps with just time and water given a standard U.S. code piping rise of 0.25 ft/ft. Sink-to-sink or retrograde transmission may explain the recurrence of pathogen colonization following intervention strategies like disinfection or replacement of plumbing . . Sink 3 was lowest on the slope in the drain line see Fig. 4 with arguably the most opportunity for reflux and retrograde wetting. Sink 1, on the other hand, was farthest away from the source sink 5 , and its P-trap had the greatest incline in the drain line connecting the sinks, which could perhaps contribute to the reasons there was no GFP-expressing E. coli colonization detected in it after 7 days. There has been more investigation about microbiologic dynamics of infectious viral particles such as those of severe acute respiratory syndrome SARS and Ebola viruses through premise plumbing systems . . . . However, the microbiology, sustainability, and dynamics might be very different, although the backflow and inoculation issues could have some parallels when comparing viruses to bacteria. As Enterobacteriaceae can either multiply or remain viable for long periods of time in biofilms coating the interior of P-traps and the connected plumbing, it may not be sustainable to target any intervention limited to a single isolated sink as a source of a particular pathogen. Data from different dispersal experiments suggest that although P-traps can act as the source or the reservoir of pathogens, the physical presence of the organism in the sink bowl or colonization of the strainer is necessary for the dispersal to occur. Colonization of strainers or drains reported in earlier studies . was perhaps a result of ascending biofilm growth from the P-trap to the strainer or introduction through contaminated fluids. Many of the studies used swab samples, which likely sampled the strainer rather than P-trap water . . Once the strainer was colonized, the water from the faucet resulted in GFP-expressing E. coli dispersion in the bowl and to the surrounding surfaces of up to 30 in. The range of dispersal . . Greater dispersal near the faucet may be attributed to the specific designs of the sink bowl and faucet in this study, which determine the contact angle of water impact. This is an important finding since many sinks in hospitals are similar in design, with faucet handles representing a high-touch surface for the sink users . . It can also be concluded from the dispersion experiments that secondary and successive dispersals would likely increase the degree and the scope of dispersion. There are several limitations to this work. First the use of similar sink bowls across these sinks only examines the dispersion pattern of this particular sink design. Similarly the sink-to-sink transmission may not be applicable to all wastewater plumbing systems as the fixtures on the pipe are very close together, unlike most layouts in health care settings. However, we speculate that transmission could occur on larger systems over greater time scales, especially if heavy nutrient and contamination loads were also included. GFP-expressing E. coli is a laboratory surrogate, and the putative biofilms established in the short time frame of our experiments are unlikely to be as complex or stable as biofilms developed in a hospital wastewater system over many years. However, to address the monomicrobial dominance of the GFP-expressing E. coli added to the system, we kept the system open, and other environmental organisms were able to cocolonize in an attempt to mimic the hospital system. Another limitation was the need to add nutrients to the drain to ensure rapid and robust colonization. We are not clear how widespread the practice of disposing of dextrose-containing intravenous fluids or leftover beverages in the hand-wash sinks is; however, we have observed this practice, and anecdotally it appears to be relatively common in the United States. We also did not completely characterize the droplet sizes, nor do we demonstrate air sampling to understand if the dispersion is only droplet or if there are also aerosols that contain GFP-expressing E. coli. This would require additional testing and is planned as future work. In summary, this work for the first time better models the mechanisms of spread of multidrug-resistant pathogens arising from the sink drain and infecting patients. Droplet dispersion from the P-trap does not happen directly. Rather it is a multistage process: dispersal originates from the strainer and/or the bowl after growth of the biofilm up from the microbial reservoir of the P-trap. We also demonstrate sink-to-sink transmission via a common sanitary pipe. This work could have implications for patient safety, infection control, and interventions as well as the design of future hospital plumbing systems to eliminate this mode of transmission to vulnerable hospitalized patients. Sink gallery design. A dedicated sink gallery was set up to simulate hospital hand-washing sinks. The gallery was comprised of five sink modules assembled next to each other Fig. 4 . The five hand-wash sink stations were identical in bowl designs and dimensions and were modeled from the most common intensive care unit hand-washing sink type in the acute care hospital at the University of Virginia Medical Center. Partitions made of 24-in.-high Plexiglas sheet were installed between the sinks to prevent splatter and cross contamination. Each sink module was built with Corian integrated sink/countertops without an overflow and fitted with an 8-in. centerset 2-handle gooseneck faucet Elkay, Oak Brook, IL . The drain line Dearborn Brass-Oatey, Cleveland, OH . All of the fixtures were made of brass with chrome plating. Each of the sink P-traps was connected to a 3-in. common cast-iron pipe sloping into a T-joint leading into the building sanitary line located behind sink 3 Fig. 4 . Inoculation, growth, and establishment of GFP-expressing E. coli in sink P-traps. For the GFP-expressing E. coli strain ATCC 25922GFP , the green fluorescent protein GFP gene is contained on a plasmid that also contains an ampicillin resistance gene. A single isolated colony of GFP-expressing E. coli grown from a Ϫ80°C stock was inoculated into 5 ml tryptic soy broth TSB Becton, Dickinson and Company, Sparks, MD containing 100 g/ml ampicillin ATCC medium 2855 . The inoculum concentration and method varied for each experiment. For establishment of GFP-expressing E. coli in sink P-traps, new autoclaved P-traps were filled with 100 ml 0.1ϫ TSB and inoculated with ϳ10 3 CFU/ml GFPexpressing E. coli. Following inoculation, both ends of the P-traps were covered with perforated Parafilm Bemis, Inc., Oshkosh, WI and allowed to incubate at room temperature 22 Ϯ 2°C for 14 days to facilitate adherent bacterial growth. The medium in the P-trap was decanted and replaced with fresh 0.1ϫ TSB every 48 h. An aliquot of decanted medium and a swab sample from the inner surface of the P-trap were plated on tryptic soy agar Becton, Dickinson and Company, Sparks, MD plates containing 100 g/ml ampicillin TSA to monitor the growth of GFP-expressing E. coli in the P-traps. TSA plates were incubated overnight at 37°C, and CFU fluorescing under UV light were enumerated. All preparatory culturing of GFP-expressing E. coli took place in a separate room from the sink gallery to avoid unintentional contamination. Installation of P-traps colonized with GFP-expressing E.coli. After the 14-day incubation, P-traps were fastened into the plumbing of the sinks Fig. 5a . The remainder of the drain line was either autoclaved strainer, tailpipe, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI , maintaining at least 1 min of contact time. After the P-trap was installed, a daily regimen was followed in which 25 ml of TSB followed by 25 ml of 0.9% NaCl solution saline were added in the ratio 1:3 via the strainer Fig. 5b to mimic the potential nutrient exposure in the hospital. Sampling and enumeration of GFP-expressing E. coli. To monitor the growth of GFP-expressing E. coli in the plumbing, sampling ports were drilled along the length of the tailpiece between the P-trap and the strainer and the trap arm between the P-trap and the common line . These holes were fitted with size 00 silicone stoppers Cole-Parmer, Vernon Hills, IL Fig. 5a . Sterile cotton swabs Covidien, Mansfield, MA presoaked in saline were inserted through these sampling ports, and samples were collected by turning the swab in a circular motion on the inner surface ϳ20 cm 2 of the tailpipes. Sample swabs were pulse-vortexed in 3 ml saline, and serial dilutions were plated on TSA. The strainer, faucet aerator, and bowl surface were sampled with presoaked swabs and processed as described earlier. Sink-to-sink transmission of bacteria. To investigate sink-to-sink transmission of bacteria, a distal sink sink 5 Fig. 4 was fitted with a P-trap inoculated with GFP-expressing E. coli. The effects of different inoculum concentrations of GFP-expressing E. coli-10 3 , 10 6 , and Ͼ10 10 CFU/ml colonized for 14 days -were investigated. Identification to the species level of fluorescent and nonfluorescent colonies identified from mixed pipe cultures was performed using a matrix-assisted laser desorption-ionization MALDI -time of flight MALDI-TOF mass spectrometer Vitek-MS; bioMérieux, Durham, NC . The wastewater paths of sinks 1 to 4 were either autoclaved strainer, tailpipe, P-traps, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI . Faucets on each of the five sinks were turned on simultaneously for 1 min, supplying water at a flow rate of 8 liters/min, once every 24 h for 7 days. No additional feed to any of the sinks was added during this period of 7 days. On days 0 and 7, P-traps on each of the five sinks were unfastened, and swab samples from the P-trap were collected and processed as described earlier. Dispersion measured using fluorescent microspheres. Fluoresbrite YO carboxylate microspheres Polysciences, Inc. which had a 1-m diameter and maximum excitation and emission of 529 nm and 546 nm, respectively, were used as a tracer in the preliminary experiments to understand droplet dispersion from the hand-wash sinks. To test whether microspheres could be dispersed from below the sink strainer, a 1-ml suspension of microspheres ϳ10 10 particles was injected through a strainer attached to a Hert 4.5-in. offset drain tailpiece typically used for wheelchair-accessible sinks American Standard, model 7723018.002 Fig. 5c . The vertical distance between the strainer and microsphere suspension injected into the tailpipe was ϳ4 in. Counter space around the sink bowl was thoroughly wiped with alcohol wipes Covidien Webcol 6818; Kendall , and polyester sheets precut to appropriate shapes were placed on the counter to cover the entire sink counter and labeled according to position Fig. 6a . The faucet was turned on for 5 min at a water flow rate of 1.8 to 3.0 liters/min. Polyester sheets were harvested and immediately analyzed using a ChemiDoc MP system Bio-Rad Laboratories, Inc. with an exposure time of 5 s. Fluorescent microspheres were enumerated from the digital micrographs using the Image Lab Software Bio-Rad Laboratories, Inc. . To test whether microspheres could be dispersed from the surface of the sink bowl, the sink bowl was evenly coated with a 20-ml microsphere suspension ϳ10 10 particles/ml using a disposable swab Sage Products, Inc., Cary, IL , and the dispersion experiment was repeated following the protocol described above. To ascertain there was no nonspecific background fluorescence in the sink and/or the water from the faucet, a control using the same protocol but without the fluorescent microspheres was performed before each experiment. Dispersion measured using GFP-expressing E.coli. Dispersion using GFP-expressing E. coli was investigated in three experiments. To test whether live organisms in the P-trap could be dispersed by running water, ϳ10 10 CFU/ml GFP-expressing E. coli in saline was added to an autoclaved P-trap and fitted into the drain line that was preautoclaved strainer, tailpipe, and trap arms . Similarly, to test whether live organisms could be dispersed from the tailpieces of wheelchair-accessible sinks, a suspension of ϳ10 10 CFU/ml GFP-expressing E. coli was added with a syringe through the strainer into the Hert 4.5-ft offset drain tailpiece Fig. 5c . Just as in the microsphere dispersion experiment, the vertical distance between the strainer and GFP-expressing E. coli suspension injected into the tailpipe was ϳ4 in. We next tested whether live organisms from the surface of the sink bowl could be dispersed by running water. The sink bowl surface was evenly coated with an approximately 20-ml suspension of 10 10 CFU/ml GFP-expressing E. coli. Finally, to mimic all of these conditions, a P-trap colonized with GFP-expressing E. coli for 14 days was installed, and a nutrient regimen Fig. 5b was followed for 14 days to intentionally promote the GFP-expressing E. coli colonization in the attached tailpipe and strainer. On day 15, the dispersion experiment was performed. Before each of the GFP-expressing E. coli dispersion experiments, the counter space was thoroughly disinfected with Caviwipes-1. TSA plates were then positioned on the sink counter surrounding the bowl and an extension platform Fig. 6b . Additional plates were attached to the sink bowl, faucets, Plexiglas partitions, and faucet handles using adhesive tape. TSA plates were also placed 3 m away from the sink as negative controls. The faucet was turned on for 5 min with a water flow rate of 1.8 to 3.0 liters/min. Lids of the TSA plates were removed only during faucet operation. Swab samples from the faucet aerators before and after operation were collected and plated on TSA. Prior to each dispersion experiment, 50 ml water from the faucet was also collected, and aliquots were plated to assess for the presence of GFP-expressing E. coli in source water and ensure cross contamination of GFP-expressing E. coli had not occurred. A control dispersion experiment was also performed using the same protocol prior to GFP-expressing E. coli inoculation in each case. Dispersion per defined area CFU per square centimeter was deduced by dividing the CFU counts in the TSA plate by the surface area of the TSA plate.
What may be a likely cause of sink-to-sink spreading of pathogens in the hospital setting?	via a common sanitary pipe	[ "There have been an increasing number of reports implicating Gammaproteobacteria as often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand-washing sink lab gallery to model dispersion of green fluorescent protein GFP -expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-expressing E. coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas <30 in.", "However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas <30 in. during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient.", "We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient. IMPORTANCE Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug-resistant bacteria, which then results in hospital-acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks.", "Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic-resistant bacteria that can thrive in wastewater environments and cause infections in vulnerable patients. Text: D espite early reports . . . . .", "Text: D espite early reports . . . . . , the premise that hand-wash sink traps can act as reservoirs of bacteria that cause nosocomial infections has been frequently overlooked. There has recently been an alarming increase in sink-related outbreaks worldwide, with many reports establishing an observational link . . . . . . . . . A sink often operates as an open conduit to wastewater in a patient care area that is often in the same room as the patient. Health care establishments often invest in desperate interventions to deal with nosocomial outbreaks. The preferred method for addressing most of the environmentrelated transmission is to employ enhanced cleaning using chemical and physical agents . .", "The preferred method for addressing most of the environmentrelated transmission is to employ enhanced cleaning using chemical and physical agents . . Unfortunately, routine approaches are inefficient in completely eliminating drug-resistant Gammaproteobacteria in an inaccessible microbiologically active area such as a sink trap 6, . . . . . . The wet, humid, and relatively protected environment in a sink trap favors the formation of rich stable microbial communities . . These communities will be exposed to liquids and waste that are discarded in a sink and may include antimicrobials, discarded beverages, soap, presumably pathogenic bacteria from health care workers' hands, and other items.", ". These communities will be exposed to liquids and waste that are discarded in a sink and may include antimicrobials, discarded beverages, soap, presumably pathogenic bacteria from health care workers' hands, and other items. In short, sink traps could serve as a breeding ground for opportunistic and highly antimicrobial-resistant bacteria that cannot be easily cleaned or removed . . . . . . . There are many reports of a genetic association between pathogens found in sink traps and those found in patients . . However, surprisingly little work has been done to understand the microscale transmission dynamics.", ". However, surprisingly little work has been done to understand the microscale transmission dynamics. It was previously demonstrated using a suspension of fluorescent particles Glo Germ; Glo Germ Co., Moab, UT that material injected into the P-trap gets dispersed around a hand-washing sink . . This result however has not been replicated hitherto in the follow-up studies. Dispersion has never been investigated with living organisms.", ". This result however has not been replicated hitherto in the follow-up studies. Dispersion has never been investigated with living organisms. Ultimately, many details remain unaddressed surrounding the spread of Enterobacteriaceae in sink-trap wastewater systems: i can organisms grow retrograde from the P-trap water to the sink strainer, ii can organisms spread from one sink to another along the internal surfaces of pipes with shared drainage systems, and iii which portion of a colonized drain pipe results in dispersion into the sink bowl during a hand-washing event? We aim to better understand the dispersion dynamics of Gammaproteobacteria living in the wastewater of a sink strainer and P-trap into an area where patients and health care workers could be exposed. To study this dynamic, we used a surrogate organism that could be easily tracked while remaining in the Enterobacteriaceae family, where some of the most concerning threats in antimicrobial resistance are developing .", "We aim to better understand the dispersion dynamics of Gammaproteobacteria living in the wastewater of a sink strainer and P-trap into an area where patients and health care workers could be exposed. To study this dynamic, we used a surrogate organism that could be easily tracked while remaining in the Enterobacteriaceae family, where some of the most concerning threats in antimicrobial resistance are developing . . Growth and colonization of GFP-expressing E. coli in the P-trap. In the first 14 days following the installation of the P-trap with established green fluorescent protein GFP -expressing Escherichia coli and just water running from the faucet, GFPexpressing E. coli was not detected in the tailpipe beyond 1.5 in. above the liquid level in the P-trap.", "In the first 14 days following the installation of the P-trap with established green fluorescent protein GFP -expressing Escherichia coli and just water running from the faucet, GFPexpressing E. coli was not detected in the tailpipe beyond 1.5 in. above the liquid level in the P-trap. GFP-expressing E. coli, however, was found to be viable in the P-trap without any nutrients added. A nutrient regimen was then instituted to understand the influence of nutrients on mobility and upward growth. The addition of tryptic soy broth TSB promoted GFP-expressing E. coli growth as early as day 1, with growth observed in the tailpipe 2 in. above the liquid surface in the P-trap Table 1 .", "The addition of tryptic soy broth TSB promoted GFP-expressing E. coli growth as early as day 1, with growth observed in the tailpipe 2 in. above the liquid surface in the P-trap Table 1 . On day 7, the strainer ϳ8 in. above the liquid in the P-trap was found to be colonized with GFP-expressing E. coli. This translates to an average growth rate of 1 in./day along the length of the tailpipe with the addition of nutrients and without faucet operation. GFP-expressing E. coli was not detected in the faucet water. Sink-to-sink transmission of bacteria.", "GFP-expressing E. coli was not detected in the faucet water. Sink-to-sink transmission of bacteria. In these experiments, a flanking sink sink 5 was the only P-trap inoculated with GFP-expressing E. coli and therefore was the sole source for transmission to the connected sinks. Starting with a lower inoculum concentration 10 3 CFU/ml in sink 5, on day 7, GFP-expressing E. coli was detected in the sink 2 and sink 3 P-traps Fig. 1a . With inoculum concentrations of 10 6 CFU/ml and Ͼ10 10 CFU/ml in sink 5, all of the sink P-traps in the sink gallery with the exception of sink 1 were found to be colonized with GFP-expressing E. coli after 7 days Fig.", "1a . With inoculum concentrations of 10 6 CFU/ml and Ͼ10 10 CFU/ml in sink 5, all of the sink P-traps in the sink gallery with the exception of sink 1 were found to be colonized with GFP-expressing E. coli after 7 days Fig. 1b and c . Faucet water and aerators tested negative for GFP-expressing E. coli. Irrespective of the starting inoculum concentration, on day 7 the highest level of colonization was recorded in the sink 3 P-trap. After day 7, when the nutrient regimen described previously was followed for an additional 7 days in each of the sinks in the sink gallery with an inoculum concentration of Ͼ10 10 CFU/ml, GFP-expressing E. coli was detected in the strainers of sinks 2 and 3 on day 14.", "Irrespective of the starting inoculum concentration, on day 7 the highest level of colonization was recorded in the sink 3 P-trap. After day 7, when the nutrient regimen described previously was followed for an additional 7 days in each of the sinks in the sink gallery with an inoculum concentration of Ͼ10 10 CFU/ml, GFP-expressing E. coli was detected in the strainers of sinks 2 and 3 on day 14. This finding validated the upward growth and growth rate in the tailpipe when nutrients were added. Nonfluorescent colonies were occasionally observed in the P-trap water samples collected from the sinks, which were subsequently identified to be Pseudomonas sp. or Stenotrophomonas maltophilia, and fluorescent colonies were confirmed to be E. coli. Dispersion of microspheres from sinks.", "or Stenotrophomonas maltophilia, and fluorescent colonies were confirmed to be E. coli. Dispersion of microspheres from sinks. In the first dispersion experiment, when fluorescent microspheres were inoculated into the offset drain tailpiece only 4 in. below the strainer, no microspheres were detected on the polyester sheets placed on the counter space. However, when the sink bowl was coated with the microspheres, polyester sheets overlaid on the counter space captured the dispersed microspheres caused by the faucet operation. Dispersion was observed on almost all zones of the sink counter space Fig. 2 .", "Dispersion was observed on almost all zones of the sink counter space Fig. 2 . Relatively higher levels of dispersion were observed along the major and minor axes of the elliptical sink bowl zones 2, 5, 6, 9, 11, and 12 . Anterior corners of the sink counter space zones 4 and 7 , which were most distant from the impact of water in the sink bowl, received the lowest dispersion. Dispersion of GFP-expressing E. coli from sinks. Initially the P-trap alone was inoculated with GFP-expressing E. coli and carefully installed, keeping the tailpipe and strainer free of GFP-expressing E. coli before operating the faucets.", "Dispersion of GFP-expressing E. coli from sinks. Initially the P-trap alone was inoculated with GFP-expressing E. coli and carefully installed, keeping the tailpipe and strainer free of GFP-expressing E. coli before operating the faucets. No fluorescent CFU were observed on the plates placed on the counter or attached to the bowl surface after faucet operation. Similarly, no fluorescent CFU were detected when GFPexpressing E. coli was inoculated into the offset drain tailpiece only 4 in. below the strainer. Interestingly, when there was conspicuous water backup over the strainer as a result of a higher water flow rate from the faucet than the drainage rate from the P-trap, dispersal was detected on the plates attached to the bowl surface.", "below the strainer. Interestingly, when there was conspicuous water backup over the strainer as a result of a higher water flow rate from the faucet than the drainage rate from the P-trap, dispersal was detected on the plates attached to the bowl surface. The dispersion pattern recorded when the sink bowl was coated with GFPexpressing E. coli was comparable to the pattern recorded when the sink bowl was coated with fluorescent microspheres Fig. 2 . Dispersion was significantly higher along the axes zones 6, 9, 11, and 12 and lower at the corners of the sink counter space zones 4, 7, and 10 . In contrast, dispersion of GFP-expressing E. coli caused by the faucet operation was much more extensive when the strainer was allowed to be colonized with GFPexpressing E. coli prior to the dispersion experiment.", "Dispersion was significantly higher along the axes zones 6, 9, 11, and 12 and lower at the corners of the sink counter space zones 4, 7, and 10 . In contrast, dispersion of GFP-expressing E. coli caused by the faucet operation was much more extensive when the strainer was allowed to be colonized with GFPexpressing E. coli prior to the dispersion experiment. In addition to the sink counter space, we measured dispersion to the sink bowl, faucet, faucet handles, splatter shields, and the extended counter surface. Dispersion of GFP-expressing E. coli was highest on the plates attached to the sink bowl Fig. 3b . Further, dispersion was greater along the Ϫ\" and \"ϩ\" denote the absence and presence of GFP-expressing E. coli, respectively.", "3b . Further, dispersion was greater along the Ϫ\" and \"ϩ\" denote the absence and presence of GFP-expressing E. coli, respectively. minor axis of the sink bowl Fig. 3b , zones B3, B4, and B10 than along the major axis of the sink bowl, associated with a shorter distance from the strike point of the faucet water to the bowl along this axis. The next highest CFU count from the dispersal was recorded on the counter area near the faucets Fig. 3a , zones 12 and 11 . A similar pattern of higher dispersion near the faucets and lower dispersion at the corners of the counter space Fig.", "3a , zones 12 and 11 . A similar pattern of higher dispersion near the faucets and lower dispersion at the corners of the counter space Fig. 3a , zones 4, 7, and 10 was also observed using microspheres. Dispersion was also recorded in other zones of the counter space, on the Plexiglas splatter shields, faucets, faucet handles, and extended surface Fig. 3c . There were no GFP-expressing E. coli CFU recorded on plates placed beyond 30 in. from the strainer, demarcating the range of dispersion under these experimental conditions. Table 2 gives a summary of the total distribution loads recorded using fluorescent microspheres and GFP-expressing E. coli across each experiment.", "from the strainer, demarcating the range of dispersion under these experimental conditions. Table 2 gives a summary of the total distribution loads recorded using fluorescent microspheres and GFP-expressing E. coli across each experiment. The loads of dispersion on the sink counter were comparable when the sink bowl was coated with microspheres or GFP-expressing E. coli before the faucet operation. Although the dispersion load on the sink counter was lower when the sink strainer was colonized, it is interesting to note that the sink bowl received the highest dispersion. To mimic dispersion in a hospital setting, we first investigated whether GFPexpressing E. coli would establish consistent colonization in a sink trap as many other Gammaproteobacteria implicated in nosocomial outbreaks have done . .", "To mimic dispersion in a hospital setting, we first investigated whether GFPexpressing E. coli would establish consistent colonization in a sink trap as many other Gammaproteobacteria implicated in nosocomial outbreaks have done . . Many recent reports demonstrate that P-traps become colonized with highly consequential Gammaproteobacteria, which then results in nosocomial transmission . . The retained water in a sink P-trap is present to provide a water barrier to prevent off-gassing of sewer smell, but it may inadvertently provide favorable conditions for pathogenic and opportunistic antibiotic-resistant microorganisms to survive and develop resilient biofilms . . However, the mechanism of dispersal of the bacteria in the P-trap to patients or the surrounding health care area had not been fully elucidated.", ". However, the mechanism of dispersal of the bacteria in the P-trap to patients or the surrounding health care area had not been fully elucidated. We began with the hypothesis that the bacteria originate from the P-trap via droplet creation when the water from the faucet hits the P-trap water, thus contaminating the sink bowl and the surrounding area. The finding supporting this theory had been previously reported using Glo Germ particles . . However, in the present study with careful attention to avoid strainer and tail piece contamination, the dispersal directly from the sink P-trap with either microspheres or GFP-expressing E. coli could not be reproduced as previously reported . .", "However, in the present study with careful attention to avoid strainer and tail piece contamination, the dispersal directly from the sink P-trap with either microspheres or GFP-expressing E. coli could not be reproduced as previously reported . . Rather this work demonstrates a different, more staged mode of transmission from a P-trap reservoir to the sink and surrounding environment. GFP-expressing E. coli in the P-trap alone was sustained for 14 days but did not grow or mobilize up the tailpipe to the strainer with just intermittent water exposure. However, when nutrients were subsequently added to the system, the organisms rapidly grew up the tailpipe to the strainer at approximately an inch per day. In a real-world setting, motility of bacteria inside the tailpipe is restricted to relatively sporadic and brief wetting events in which swimming is an opportunity to colonize new surfaces.", "However, when nutrients were subsequently added to the system, the organisms rapidly grew up the tailpipe to the strainer at approximately an inch per day. In a real-world setting, motility of bacteria inside the tailpipe is restricted to relatively sporadic and brief wetting events in which swimming is an opportunity to colonize new surfaces. It is assumed that once established, the biofilm promotes the upward growth of GFP-expressing E. coli in the tailpipe at an accelerated rate. The nutrient regimen that promoted colonization in our model reflects our and others' observations of items commonly disposed of in hospital sinks intravenous fluids, feeding supplements, and leftover beverages . . Transmission of bacteria between sinks via a common pipe was a key finding in this study as this highlights the concept that premise plumbing may be a more continuous system with shared microbiology than a single isolated sink.", ". Transmission of bacteria between sinks via a common pipe was a key finding in this study as this highlights the concept that premise plumbing may be a more continuous system with shared microbiology than a single isolated sink. The sink gallery used in this study provided a unique in situ advantage to investigate sink-to-sink transmission of bacteria through common drains. The two possible mechanisms for P-trap strainers becoming colonized are seeding of organisms from above and retrograde spread of organisms along common pipes in a hospital wastewater infrastructure. Here we demonstrate that it is possible for GFP-expressing E. coli to contaminate adjacent P-traps with just time and water given a standard U.S. code piping rise of 0.25 ft/ft. Sink-to-sink or retrograde transmission may explain the recurrence of pathogen colonization following intervention strategies like disinfection or replacement of plumbing .", "Here we demonstrate that it is possible for GFP-expressing E. coli to contaminate adjacent P-traps with just time and water given a standard U.S. code piping rise of 0.25 ft/ft. Sink-to-sink or retrograde transmission may explain the recurrence of pathogen colonization following intervention strategies like disinfection or replacement of plumbing . . Sink 3 was lowest on the slope in the drain line see Fig. 4 with arguably the most opportunity for reflux and retrograde wetting. Sink 1, on the other hand, was farthest away from the source sink 5 , and its P-trap had the greatest incline in the drain line connecting the sinks, which could perhaps contribute to the reasons there was no GFP-expressing E. coli colonization detected in it after 7 days.", "4 with arguably the most opportunity for reflux and retrograde wetting. Sink 1, on the other hand, was farthest away from the source sink 5 , and its P-trap had the greatest incline in the drain line connecting the sinks, which could perhaps contribute to the reasons there was no GFP-expressing E. coli colonization detected in it after 7 days. There has been more investigation about microbiologic dynamics of infectious viral particles such as those of severe acute respiratory syndrome SARS and Ebola viruses through premise plumbing systems . . . . However, the microbiology, sustainability, and dynamics might be very different, although the backflow and inoculation issues could have some parallels when comparing viruses to bacteria.", ". . However, the microbiology, sustainability, and dynamics might be very different, although the backflow and inoculation issues could have some parallels when comparing viruses to bacteria. As Enterobacteriaceae can either multiply or remain viable for long periods of time in biofilms coating the interior of P-traps and the connected plumbing, it may not be sustainable to target any intervention limited to a single isolated sink as a source of a particular pathogen. Data from different dispersal experiments suggest that although P-traps can act as the source or the reservoir of pathogens, the physical presence of the organism in the sink bowl or colonization of the strainer is necessary for the dispersal to occur. Colonization of strainers or drains reported in earlier studies .", "Data from different dispersal experiments suggest that although P-traps can act as the source or the reservoir of pathogens, the physical presence of the organism in the sink bowl or colonization of the strainer is necessary for the dispersal to occur. Colonization of strainers or drains reported in earlier studies . was perhaps a result of ascending biofilm growth from the P-trap to the strainer or introduction through contaminated fluids. Many of the studies used swab samples, which likely sampled the strainer rather than P-trap water . . Once the strainer was colonized, the water from the faucet resulted in GFP-expressing E. coli dispersion in the bowl and to the surrounding surfaces of up to 30 in.", ". Once the strainer was colonized, the water from the faucet resulted in GFP-expressing E. coli dispersion in the bowl and to the surrounding surfaces of up to 30 in. The range of dispersal . . Greater dispersal near the faucet may be attributed to the specific designs of the sink bowl and faucet in this study, which determine the contact angle of water impact. This is an important finding since many sinks in hospitals are similar in design, with faucet handles representing a high-touch surface for the sink users . . It can also be concluded from the dispersion experiments that secondary and successive dispersals would likely increase the degree and the scope of dispersion.", ". It can also be concluded from the dispersion experiments that secondary and successive dispersals would likely increase the degree and the scope of dispersion. There are several limitations to this work. First the use of similar sink bowls across these sinks only examines the dispersion pattern of this particular sink design. Similarly the sink-to-sink transmission may not be applicable to all wastewater plumbing systems as the fixtures on the pipe are very close together, unlike most layouts in health care settings. However, we speculate that transmission could occur on larger systems over greater time scales, especially if heavy nutrient and contamination loads were also included.", "Similarly the sink-to-sink transmission may not be applicable to all wastewater plumbing systems as the fixtures on the pipe are very close together, unlike most layouts in health care settings. However, we speculate that transmission could occur on larger systems over greater time scales, especially if heavy nutrient and contamination loads were also included. GFP-expressing E. coli is a laboratory surrogate, and the putative biofilms established in the short time frame of our experiments are unlikely to be as complex or stable as biofilms developed in a hospital wastewater system over many years. However, to address the monomicrobial dominance of the GFP-expressing E. coli added to the system, we kept the system open, and other environmental organisms were able to cocolonize in an attempt to mimic the hospital system. Another limitation was the need to add nutrients to the drain to ensure rapid and robust colonization. We are not clear how widespread the practice of disposing of dextrose-containing intravenous fluids or leftover beverages in the hand-wash sinks is; however, we have observed this practice, and anecdotally it appears to be relatively common in the United States.", "Another limitation was the need to add nutrients to the drain to ensure rapid and robust colonization. We are not clear how widespread the practice of disposing of dextrose-containing intravenous fluids or leftover beverages in the hand-wash sinks is; however, we have observed this practice, and anecdotally it appears to be relatively common in the United States. We also did not completely characterize the droplet sizes, nor do we demonstrate air sampling to understand if the dispersion is only droplet or if there are also aerosols that contain GFP-expressing E. coli. This would require additional testing and is planned as future work. In summary, this work for the first time better models the mechanisms of spread of multidrug-resistant pathogens arising from the sink drain and infecting patients. Droplet dispersion from the P-trap does not happen directly.", "In summary, this work for the first time better models the mechanisms of spread of multidrug-resistant pathogens arising from the sink drain and infecting patients. Droplet dispersion from the P-trap does not happen directly. Rather it is a multistage process: dispersal originates from the strainer and/or the bowl after growth of the biofilm up from the microbial reservoir of the P-trap. We also demonstrate sink-to-sink transmission via a common sanitary pipe. This work could have implications for patient safety, infection control, and interventions as well as the design of future hospital plumbing systems to eliminate this mode of transmission to vulnerable hospitalized patients. Sink gallery design.", "This work could have implications for patient safety, infection control, and interventions as well as the design of future hospital plumbing systems to eliminate this mode of transmission to vulnerable hospitalized patients. Sink gallery design. A dedicated sink gallery was set up to simulate hospital hand-washing sinks. The gallery was comprised of five sink modules assembled next to each other Fig. 4 . The five hand-wash sink stations were identical in bowl designs and dimensions and were modeled from the most common intensive care unit hand-washing sink type in the acute care hospital at the University of Virginia Medical Center. Partitions made of 24-in.-high Plexiglas sheet were installed between the sinks to prevent splatter and cross contamination.", "The five hand-wash sink stations were identical in bowl designs and dimensions and were modeled from the most common intensive care unit hand-washing sink type in the acute care hospital at the University of Virginia Medical Center. Partitions made of 24-in.-high Plexiglas sheet were installed between the sinks to prevent splatter and cross contamination. Each sink module was built with Corian integrated sink/countertops without an overflow and fitted with an 8-in. centerset 2-handle gooseneck faucet Elkay, Oak Brook, IL . The drain line Dearborn Brass-Oatey, Cleveland, OH . All of the fixtures were made of brass with chrome plating. Each of the sink P-traps was connected to a 3-in.", "All of the fixtures were made of brass with chrome plating. Each of the sink P-traps was connected to a 3-in. common cast-iron pipe sloping into a T-joint leading into the building sanitary line located behind sink 3 Fig. 4 . Inoculation, growth, and establishment of GFP-expressing E. coli in sink P-traps. For the GFP-expressing E. coli strain ATCC 25922GFP , the green fluorescent protein GFP gene is contained on a plasmid that also contains an ampicillin resistance gene. A single isolated colony of GFP-expressing E. coli grown from a Ϫ80°C stock was inoculated into 5 ml tryptic soy broth TSB Becton, Dickinson and Company, Sparks, MD containing 100 g/ml ampicillin ATCC medium 2855 .", "For the GFP-expressing E. coli strain ATCC 25922GFP , the green fluorescent protein GFP gene is contained on a plasmid that also contains an ampicillin resistance gene. A single isolated colony of GFP-expressing E. coli grown from a Ϫ80°C stock was inoculated into 5 ml tryptic soy broth TSB Becton, Dickinson and Company, Sparks, MD containing 100 g/ml ampicillin ATCC medium 2855 . The inoculum concentration and method varied for each experiment. For establishment of GFP-expressing E. coli in sink P-traps, new autoclaved P-traps were filled with 100 ml 0.1ϫ TSB and inoculated with ϳ10 3 CFU/ml GFPexpressing E. coli. Following inoculation, both ends of the P-traps were covered with perforated Parafilm Bemis, Inc., Oshkosh, WI and allowed to incubate at room temperature 22 Ϯ 2°C for 14 days to facilitate adherent bacterial growth. The medium in the P-trap was decanted and replaced with fresh 0.1ϫ TSB every 48 h. An aliquot of decanted medium and a swab sample from the inner surface of the P-trap were plated on tryptic soy agar Becton, Dickinson and Company, Sparks, MD plates containing 100 g/ml ampicillin TSA to monitor the growth of GFP-expressing E. coli in the P-traps.", "Following inoculation, both ends of the P-traps were covered with perforated Parafilm Bemis, Inc., Oshkosh, WI and allowed to incubate at room temperature 22 Ϯ 2°C for 14 days to facilitate adherent bacterial growth. The medium in the P-trap was decanted and replaced with fresh 0.1ϫ TSB every 48 h. An aliquot of decanted medium and a swab sample from the inner surface of the P-trap were plated on tryptic soy agar Becton, Dickinson and Company, Sparks, MD plates containing 100 g/ml ampicillin TSA to monitor the growth of GFP-expressing E. coli in the P-traps. TSA plates were incubated overnight at 37°C, and CFU fluorescing under UV light were enumerated. All preparatory culturing of GFP-expressing E. coli took place in a separate room from the sink gallery to avoid unintentional contamination. Installation of P-traps colonized with GFP-expressing E.coli. After the 14-day incubation, P-traps were fastened into the plumbing of the sinks Fig.", "Installation of P-traps colonized with GFP-expressing E.coli. After the 14-day incubation, P-traps were fastened into the plumbing of the sinks Fig. 5a . The remainder of the drain line was either autoclaved strainer, tailpipe, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI , maintaining at least 1 min of contact time. After the P-trap was installed, a daily regimen was followed in which 25 ml of TSB followed by 25 ml of 0.9% NaCl solution saline were added in the ratio 1:3 via the strainer Fig. 5b to mimic the potential nutrient exposure in the hospital.", "After the P-trap was installed, a daily regimen was followed in which 25 ml of TSB followed by 25 ml of 0.9% NaCl solution saline were added in the ratio 1:3 via the strainer Fig. 5b to mimic the potential nutrient exposure in the hospital. Sampling and enumeration of GFP-expressing E. coli. To monitor the growth of GFP-expressing E. coli in the plumbing, sampling ports were drilled along the length of the tailpiece between the P-trap and the strainer and the trap arm between the P-trap and the common line . These holes were fitted with size 00 silicone stoppers Cole-Parmer, Vernon Hills, IL Fig. 5a .", "These holes were fitted with size 00 silicone stoppers Cole-Parmer, Vernon Hills, IL Fig. 5a . Sterile cotton swabs Covidien, Mansfield, MA presoaked in saline were inserted through these sampling ports, and samples were collected by turning the swab in a circular motion on the inner surface ϳ20 cm 2 of the tailpipes. Sample swabs were pulse-vortexed in 3 ml saline, and serial dilutions were plated on TSA. The strainer, faucet aerator, and bowl surface were sampled with presoaked swabs and processed as described earlier. Sink-to-sink transmission of bacteria. To investigate sink-to-sink transmission of bacteria, a distal sink sink 5 Fig.", "Sink-to-sink transmission of bacteria. To investigate sink-to-sink transmission of bacteria, a distal sink sink 5 Fig. 4 was fitted with a P-trap inoculated with GFP-expressing E. coli. The effects of different inoculum concentrations of GFP-expressing E. coli-10 3 , 10 6 , and Ͼ10 10 CFU/ml colonized for 14 days -were investigated. Identification to the species level of fluorescent and nonfluorescent colonies identified from mixed pipe cultures was performed using a matrix-assisted laser desorption-ionization MALDI -time of flight MALDI-TOF mass spectrometer Vitek-MS; bioMérieux, Durham, NC . The wastewater paths of sinks 1 to 4 were either autoclaved strainer, tailpipe, P-traps, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI .", "Identification to the species level of fluorescent and nonfluorescent colonies identified from mixed pipe cultures was performed using a matrix-assisted laser desorption-ionization MALDI -time of flight MALDI-TOF mass spectrometer Vitek-MS; bioMérieux, Durham, NC . The wastewater paths of sinks 1 to 4 were either autoclaved strainer, tailpipe, P-traps, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI . Faucets on each of the five sinks were turned on simultaneously for 1 min, supplying water at a flow rate of 8 liters/min, once every 24 h for 7 days. No additional feed to any of the sinks was added during this period of 7 days. On days 0 and 7, P-traps on each of the five sinks were unfastened, and swab samples from the P-trap were collected and processed as described earlier. Dispersion measured using fluorescent microspheres.", "On days 0 and 7, P-traps on each of the five sinks were unfastened, and swab samples from the P-trap were collected and processed as described earlier. Dispersion measured using fluorescent microspheres. Fluoresbrite YO carboxylate microspheres Polysciences, Inc. which had a 1-m diameter and maximum excitation and emission of 529 nm and 546 nm, respectively, were used as a tracer in the preliminary experiments to understand droplet dispersion from the hand-wash sinks. To test whether microspheres could be dispersed from below the sink strainer, a 1-ml suspension of microspheres ϳ10 10 particles was injected through a strainer attached to a Hert 4.5-in. offset drain tailpiece typically used for wheelchair-accessible sinks American Standard, model 7723018.002 Fig. 5c .", "offset drain tailpiece typically used for wheelchair-accessible sinks American Standard, model 7723018.002 Fig. 5c . The vertical distance between the strainer and microsphere suspension injected into the tailpipe was ϳ4 in. Counter space around the sink bowl was thoroughly wiped with alcohol wipes Covidien Webcol 6818; Kendall , and polyester sheets precut to appropriate shapes were placed on the counter to cover the entire sink counter and labeled according to position Fig. 6a . The faucet was turned on for 5 min at a water flow rate of 1.8 to 3.0 liters/min.", "6a . The faucet was turned on for 5 min at a water flow rate of 1.8 to 3.0 liters/min. Polyester sheets were harvested and immediately analyzed using a ChemiDoc MP system Bio-Rad Laboratories, Inc. with an exposure time of 5 s. Fluorescent microspheres were enumerated from the digital micrographs using the Image Lab Software Bio-Rad Laboratories, Inc. . To test whether microspheres could be dispersed from the surface of the sink bowl, the sink bowl was evenly coated with a 20-ml microsphere suspension ϳ10 10 particles/ml using a disposable swab Sage Products, Inc., Cary, IL , and the dispersion experiment was repeated following the protocol described above. To ascertain there was no nonspecific background fluorescence in the sink and/or the water from the faucet, a control using the same protocol but without the fluorescent microspheres was performed before each experiment. Dispersion measured using GFP-expressing E.coli.", "To ascertain there was no nonspecific background fluorescence in the sink and/or the water from the faucet, a control using the same protocol but without the fluorescent microspheres was performed before each experiment. Dispersion measured using GFP-expressing E.coli. Dispersion using GFP-expressing E. coli was investigated in three experiments. To test whether live organisms in the P-trap could be dispersed by running water, ϳ10 10 CFU/ml GFP-expressing E. coli in saline was added to an autoclaved P-trap and fitted into the drain line that was preautoclaved strainer, tailpipe, and trap arms . Similarly, to test whether live organisms could be dispersed from the tailpieces of wheelchair-accessible sinks, a suspension of ϳ10 10 CFU/ml GFP-expressing E. coli was added with a syringe through the strainer into the Hert 4.5-ft offset drain tailpiece Fig. 5c .", "Similarly, to test whether live organisms could be dispersed from the tailpieces of wheelchair-accessible sinks, a suspension of ϳ10 10 CFU/ml GFP-expressing E. coli was added with a syringe through the strainer into the Hert 4.5-ft offset drain tailpiece Fig. 5c . Just as in the microsphere dispersion experiment, the vertical distance between the strainer and GFP-expressing E. coli suspension injected into the tailpipe was ϳ4 in. We next tested whether live organisms from the surface of the sink bowl could be dispersed by running water. The sink bowl surface was evenly coated with an approximately 20-ml suspension of 10 10 CFU/ml GFP-expressing E. coli. Finally, to mimic all of these conditions, a P-trap colonized with GFP-expressing E. coli for 14 days was installed, and a nutrient regimen Fig.", "The sink bowl surface was evenly coated with an approximately 20-ml suspension of 10 10 CFU/ml GFP-expressing E. coli. Finally, to mimic all of these conditions, a P-trap colonized with GFP-expressing E. coli for 14 days was installed, and a nutrient regimen Fig. 5b was followed for 14 days to intentionally promote the GFP-expressing E. coli colonization in the attached tailpipe and strainer. On day 15, the dispersion experiment was performed. Before each of the GFP-expressing E. coli dispersion experiments, the counter space was thoroughly disinfected with Caviwipes-1. TSA plates were then positioned on the sink counter surrounding the bowl and an extension platform Fig. 6b .", "TSA plates were then positioned on the sink counter surrounding the bowl and an extension platform Fig. 6b . Additional plates were attached to the sink bowl, faucets, Plexiglas partitions, and faucet handles using adhesive tape. TSA plates were also placed 3 m away from the sink as negative controls. The faucet was turned on for 5 min with a water flow rate of 1.8 to 3.0 liters/min. Lids of the TSA plates were removed only during faucet operation. Swab samples from the faucet aerators before and after operation were collected and plated on TSA.", "Lids of the TSA plates were removed only during faucet operation. Swab samples from the faucet aerators before and after operation were collected and plated on TSA. Prior to each dispersion experiment, 50 ml water from the faucet was also collected, and aliquots were plated to assess for the presence of GFP-expressing E. coli in source water and ensure cross contamination of GFP-expressing E. coli had not occurred. A control dispersion experiment was also performed using the same protocol prior to GFP-expressing E. coli inoculation in each case. Dispersion per defined area CFU per square centimeter was deduced by dividing the CFU counts in the TSA plate by the surface area of the TSA plate." ]	2,585	545	There have been an increasing number of reports implicating Gammaproteobacteria as often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand-washing sink lab gallery to model dispersion of green fluorescent protein GFP -expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-expressing E. coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas <30 in. during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient. IMPORTANCE Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug-resistant bacteria, which then results in hospital-acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic-resistant bacteria that can thrive in wastewater environments and cause infections in vulnerable patients. Text: D espite early reports . . . . . , the premise that hand-wash sink traps can act as reservoirs of bacteria that cause nosocomial infections has been frequently overlooked. There has recently been an alarming increase in sink-related outbreaks worldwide, with many reports establishing an observational link . . . . . . . . . A sink often operates as an open conduit to wastewater in a patient care area that is often in the same room as the patient. Health care establishments often invest in desperate interventions to deal with nosocomial outbreaks. The preferred method for addressing most of the environmentrelated transmission is to employ enhanced cleaning using chemical and physical agents . . Unfortunately, routine approaches are inefficient in completely eliminating drug-resistant Gammaproteobacteria in an inaccessible microbiologically active area such as a sink trap 6, . . . . . . The wet, humid, and relatively protected environment in a sink trap favors the formation of rich stable microbial communities . . These communities will be exposed to liquids and waste that are discarded in a sink and may include antimicrobials, discarded beverages, soap, presumably pathogenic bacteria from health care workers' hands, and other items. In short, sink traps could serve as a breeding ground for opportunistic and highly antimicrobial-resistant bacteria that cannot be easily cleaned or removed . . . . . . . There are many reports of a genetic association between pathogens found in sink traps and those found in patients . . However, surprisingly little work has been done to understand the microscale transmission dynamics. It was previously demonstrated using a suspension of fluorescent particles Glo Germ; Glo Germ Co., Moab, UT that material injected into the P-trap gets dispersed around a hand-washing sink . . This result however has not been replicated hitherto in the follow-up studies. Dispersion has never been investigated with living organisms. Ultimately, many details remain unaddressed surrounding the spread of Enterobacteriaceae in sink-trap wastewater systems: i can organisms grow retrograde from the P-trap water to the sink strainer, ii can organisms spread from one sink to another along the internal surfaces of pipes with shared drainage systems, and iii which portion of a colonized drain pipe results in dispersion into the sink bowl during a hand-washing event? We aim to better understand the dispersion dynamics of Gammaproteobacteria living in the wastewater of a sink strainer and P-trap into an area where patients and health care workers could be exposed. To study this dynamic, we used a surrogate organism that could be easily tracked while remaining in the Enterobacteriaceae family, where some of the most concerning threats in antimicrobial resistance are developing . . Growth and colonization of GFP-expressing E. coli in the P-trap. In the first 14 days following the installation of the P-trap with established green fluorescent protein GFP -expressing Escherichia coli and just water running from the faucet, GFPexpressing E. coli was not detected in the tailpipe beyond 1.5 in. above the liquid level in the P-trap. GFP-expressing E. coli, however, was found to be viable in the P-trap without any nutrients added. A nutrient regimen was then instituted to understand the influence of nutrients on mobility and upward growth. The addition of tryptic soy broth TSB promoted GFP-expressing E. coli growth as early as day 1, with growth observed in the tailpipe 2 in. above the liquid surface in the P-trap Table 1 . On day 7, the strainer ϳ8 in. above the liquid in the P-trap was found to be colonized with GFP-expressing E. coli. This translates to an average growth rate of 1 in./day along the length of the tailpipe with the addition of nutrients and without faucet operation. GFP-expressing E. coli was not detected in the faucet water. Sink-to-sink transmission of bacteria. In these experiments, a flanking sink sink 5 was the only P-trap inoculated with GFP-expressing E. coli and therefore was the sole source for transmission to the connected sinks. Starting with a lower inoculum concentration 10 3 CFU/ml in sink 5, on day 7, GFP-expressing E. coli was detected in the sink 2 and sink 3 P-traps Fig. 1a . With inoculum concentrations of 10 6 CFU/ml and Ͼ10 10 CFU/ml in sink 5, all of the sink P-traps in the sink gallery with the exception of sink 1 were found to be colonized with GFP-expressing E. coli after 7 days Fig. 1b and c . Faucet water and aerators tested negative for GFP-expressing E. coli. Irrespective of the starting inoculum concentration, on day 7 the highest level of colonization was recorded in the sink 3 P-trap. After day 7, when the nutrient regimen described previously was followed for an additional 7 days in each of the sinks in the sink gallery with an inoculum concentration of Ͼ10 10 CFU/ml, GFP-expressing E. coli was detected in the strainers of sinks 2 and 3 on day 14. This finding validated the upward growth and growth rate in the tailpipe when nutrients were added. Nonfluorescent colonies were occasionally observed in the P-trap water samples collected from the sinks, which were subsequently identified to be Pseudomonas sp. or Stenotrophomonas maltophilia, and fluorescent colonies were confirmed to be E. coli. Dispersion of microspheres from sinks. In the first dispersion experiment, when fluorescent microspheres were inoculated into the offset drain tailpiece only 4 in. below the strainer, no microspheres were detected on the polyester sheets placed on the counter space. However, when the sink bowl was coated with the microspheres, polyester sheets overlaid on the counter space captured the dispersed microspheres caused by the faucet operation. Dispersion was observed on almost all zones of the sink counter space Fig. 2 . Relatively higher levels of dispersion were observed along the major and minor axes of the elliptical sink bowl zones 2, 5, 6, 9, 11, and 12 . Anterior corners of the sink counter space zones 4 and 7 , which were most distant from the impact of water in the sink bowl, received the lowest dispersion. Dispersion of GFP-expressing E. coli from sinks. Initially the P-trap alone was inoculated with GFP-expressing E. coli and carefully installed, keeping the tailpipe and strainer free of GFP-expressing E. coli before operating the faucets. No fluorescent CFU were observed on the plates placed on the counter or attached to the bowl surface after faucet operation. Similarly, no fluorescent CFU were detected when GFPexpressing E. coli was inoculated into the offset drain tailpiece only 4 in. below the strainer. Interestingly, when there was conspicuous water backup over the strainer as a result of a higher water flow rate from the faucet than the drainage rate from the P-trap, dispersal was detected on the plates attached to the bowl surface. The dispersion pattern recorded when the sink bowl was coated with GFPexpressing E. coli was comparable to the pattern recorded when the sink bowl was coated with fluorescent microspheres Fig. 2 . Dispersion was significantly higher along the axes zones 6, 9, 11, and 12 and lower at the corners of the sink counter space zones 4, 7, and 10 . In contrast, dispersion of GFP-expressing E. coli caused by the faucet operation was much more extensive when the strainer was allowed to be colonized with GFPexpressing E. coli prior to the dispersion experiment. In addition to the sink counter space, we measured dispersion to the sink bowl, faucet, faucet handles, splatter shields, and the extended counter surface. Dispersion of GFP-expressing E. coli was highest on the plates attached to the sink bowl Fig. 3b . Further, dispersion was greater along the Ϫ" and "ϩ" denote the absence and presence of GFP-expressing E. coli, respectively. minor axis of the sink bowl Fig. 3b , zones B3, B4, and B10 than along the major axis of the sink bowl, associated with a shorter distance from the strike point of the faucet water to the bowl along this axis. The next highest CFU count from the dispersal was recorded on the counter area near the faucets Fig. 3a , zones 12 and 11 . A similar pattern of higher dispersion near the faucets and lower dispersion at the corners of the counter space Fig. 3a , zones 4, 7, and 10 was also observed using microspheres. Dispersion was also recorded in other zones of the counter space, on the Plexiglas splatter shields, faucets, faucet handles, and extended surface Fig. 3c . There were no GFP-expressing E. coli CFU recorded on plates placed beyond 30 in. from the strainer, demarcating the range of dispersion under these experimental conditions. Table 2 gives a summary of the total distribution loads recorded using fluorescent microspheres and GFP-expressing E. coli across each experiment. The loads of dispersion on the sink counter were comparable when the sink bowl was coated with microspheres or GFP-expressing E. coli before the faucet operation. Although the dispersion load on the sink counter was lower when the sink strainer was colonized, it is interesting to note that the sink bowl received the highest dispersion. To mimic dispersion in a hospital setting, we first investigated whether GFPexpressing E. coli would establish consistent colonization in a sink trap as many other Gammaproteobacteria implicated in nosocomial outbreaks have done . . Many recent reports demonstrate that P-traps become colonized with highly consequential Gammaproteobacteria, which then results in nosocomial transmission . . The retained water in a sink P-trap is present to provide a water barrier to prevent off-gassing of sewer smell, but it may inadvertently provide favorable conditions for pathogenic and opportunistic antibiotic-resistant microorganisms to survive and develop resilient biofilms . . However, the mechanism of dispersal of the bacteria in the P-trap to patients or the surrounding health care area had not been fully elucidated. We began with the hypothesis that the bacteria originate from the P-trap via droplet creation when the water from the faucet hits the P-trap water, thus contaminating the sink bowl and the surrounding area. The finding supporting this theory had been previously reported using Glo Germ particles . . However, in the present study with careful attention to avoid strainer and tail piece contamination, the dispersal directly from the sink P-trap with either microspheres or GFP-expressing E. coli could not be reproduced as previously reported . . Rather this work demonstrates a different, more staged mode of transmission from a P-trap reservoir to the sink and surrounding environment. GFP-expressing E. coli in the P-trap alone was sustained for 14 days but did not grow or mobilize up the tailpipe to the strainer with just intermittent water exposure. However, when nutrients were subsequently added to the system, the organisms rapidly grew up the tailpipe to the strainer at approximately an inch per day. In a real-world setting, motility of bacteria inside the tailpipe is restricted to relatively sporadic and brief wetting events in which swimming is an opportunity to colonize new surfaces. It is assumed that once established, the biofilm promotes the upward growth of GFP-expressing E. coli in the tailpipe at an accelerated rate. The nutrient regimen that promoted colonization in our model reflects our and others' observations of items commonly disposed of in hospital sinks intravenous fluids, feeding supplements, and leftover beverages . . Transmission of bacteria between sinks via a common pipe was a key finding in this study as this highlights the concept that premise plumbing may be a more continuous system with shared microbiology than a single isolated sink. The sink gallery used in this study provided a unique in situ advantage to investigate sink-to-sink transmission of bacteria through common drains. The two possible mechanisms for P-trap strainers becoming colonized are seeding of organisms from above and retrograde spread of organisms along common pipes in a hospital wastewater infrastructure. Here we demonstrate that it is possible for GFP-expressing E. coli to contaminate adjacent P-traps with just time and water given a standard U.S. code piping rise of 0.25 ft/ft. Sink-to-sink or retrograde transmission may explain the recurrence of pathogen colonization following intervention strategies like disinfection or replacement of plumbing . . Sink 3 was lowest on the slope in the drain line see Fig. 4 with arguably the most opportunity for reflux and retrograde wetting. Sink 1, on the other hand, was farthest away from the source sink 5 , and its P-trap had the greatest incline in the drain line connecting the sinks, which could perhaps contribute to the reasons there was no GFP-expressing E. coli colonization detected in it after 7 days. There has been more investigation about microbiologic dynamics of infectious viral particles such as those of severe acute respiratory syndrome SARS and Ebola viruses through premise plumbing systems . . . . However, the microbiology, sustainability, and dynamics might be very different, although the backflow and inoculation issues could have some parallels when comparing viruses to bacteria. As Enterobacteriaceae can either multiply or remain viable for long periods of time in biofilms coating the interior of P-traps and the connected plumbing, it may not be sustainable to target any intervention limited to a single isolated sink as a source of a particular pathogen. Data from different dispersal experiments suggest that although P-traps can act as the source or the reservoir of pathogens, the physical presence of the organism in the sink bowl or colonization of the strainer is necessary for the dispersal to occur. Colonization of strainers or drains reported in earlier studies . was perhaps a result of ascending biofilm growth from the P-trap to the strainer or introduction through contaminated fluids. Many of the studies used swab samples, which likely sampled the strainer rather than P-trap water . . Once the strainer was colonized, the water from the faucet resulted in GFP-expressing E. coli dispersion in the bowl and to the surrounding surfaces of up to 30 in. The range of dispersal . . Greater dispersal near the faucet may be attributed to the specific designs of the sink bowl and faucet in this study, which determine the contact angle of water impact. This is an important finding since many sinks in hospitals are similar in design, with faucet handles representing a high-touch surface for the sink users . . It can also be concluded from the dispersion experiments that secondary and successive dispersals would likely increase the degree and the scope of dispersion. There are several limitations to this work. First the use of similar sink bowls across these sinks only examines the dispersion pattern of this particular sink design. Similarly the sink-to-sink transmission may not be applicable to all wastewater plumbing systems as the fixtures on the pipe are very close together, unlike most layouts in health care settings. However, we speculate that transmission could occur on larger systems over greater time scales, especially if heavy nutrient and contamination loads were also included. GFP-expressing E. coli is a laboratory surrogate, and the putative biofilms established in the short time frame of our experiments are unlikely to be as complex or stable as biofilms developed in a hospital wastewater system over many years. However, to address the monomicrobial dominance of the GFP-expressing E. coli added to the system, we kept the system open, and other environmental organisms were able to cocolonize in an attempt to mimic the hospital system. Another limitation was the need to add nutrients to the drain to ensure rapid and robust colonization. We are not clear how widespread the practice of disposing of dextrose-containing intravenous fluids or leftover beverages in the hand-wash sinks is; however, we have observed this practice, and anecdotally it appears to be relatively common in the United States. We also did not completely characterize the droplet sizes, nor do we demonstrate air sampling to understand if the dispersion is only droplet or if there are also aerosols that contain GFP-expressing E. coli. This would require additional testing and is planned as future work. In summary, this work for the first time better models the mechanisms of spread of multidrug-resistant pathogens arising from the sink drain and infecting patients. Droplet dispersion from the P-trap does not happen directly. Rather it is a multistage process: dispersal originates from the strainer and/or the bowl after growth of the biofilm up from the microbial reservoir of the P-trap. We also demonstrate sink-to-sink transmission via a common sanitary pipe. This work could have implications for patient safety, infection control, and interventions as well as the design of future hospital plumbing systems to eliminate this mode of transmission to vulnerable hospitalized patients. Sink gallery design. A dedicated sink gallery was set up to simulate hospital hand-washing sinks. The gallery was comprised of five sink modules assembled next to each other Fig. 4 . The five hand-wash sink stations were identical in bowl designs and dimensions and were modeled from the most common intensive care unit hand-washing sink type in the acute care hospital at the University of Virginia Medical Center. Partitions made of 24-in.-high Plexiglas sheet were installed between the sinks to prevent splatter and cross contamination. Each sink module was built with Corian integrated sink/countertops without an overflow and fitted with an 8-in. centerset 2-handle gooseneck faucet Elkay, Oak Brook, IL . The drain line Dearborn Brass-Oatey, Cleveland, OH . All of the fixtures were made of brass with chrome plating. Each of the sink P-traps was connected to a 3-in. common cast-iron pipe sloping into a T-joint leading into the building sanitary line located behind sink 3 Fig. 4 . Inoculation, growth, and establishment of GFP-expressing E. coli in sink P-traps. For the GFP-expressing E. coli strain ATCC 25922GFP , the green fluorescent protein GFP gene is contained on a plasmid that also contains an ampicillin resistance gene. A single isolated colony of GFP-expressing E. coli grown from a Ϫ80°C stock was inoculated into 5 ml tryptic soy broth TSB Becton, Dickinson and Company, Sparks, MD containing 100 g/ml ampicillin ATCC medium 2855 . The inoculum concentration and method varied for each experiment. For establishment of GFP-expressing E. coli in sink P-traps, new autoclaved P-traps were filled with 100 ml 0.1ϫ TSB and inoculated with ϳ10 3 CFU/ml GFPexpressing E. coli. Following inoculation, both ends of the P-traps were covered with perforated Parafilm Bemis, Inc., Oshkosh, WI and allowed to incubate at room temperature 22 Ϯ 2°C for 14 days to facilitate adherent bacterial growth. The medium in the P-trap was decanted and replaced with fresh 0.1ϫ TSB every 48 h. An aliquot of decanted medium and a swab sample from the inner surface of the P-trap were plated on tryptic soy agar Becton, Dickinson and Company, Sparks, MD plates containing 100 g/ml ampicillin TSA to monitor the growth of GFP-expressing E. coli in the P-traps. TSA plates were incubated overnight at 37°C, and CFU fluorescing under UV light were enumerated. All preparatory culturing of GFP-expressing E. coli took place in a separate room from the sink gallery to avoid unintentional contamination. Installation of P-traps colonized with GFP-expressing E.coli. After the 14-day incubation, P-traps were fastened into the plumbing of the sinks Fig. 5a . The remainder of the drain line was either autoclaved strainer, tailpipe, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI , maintaining at least 1 min of contact time. After the P-trap was installed, a daily regimen was followed in which 25 ml of TSB followed by 25 ml of 0.9% NaCl solution saline were added in the ratio 1:3 via the strainer Fig. 5b to mimic the potential nutrient exposure in the hospital. Sampling and enumeration of GFP-expressing E. coli. To monitor the growth of GFP-expressing E. coli in the plumbing, sampling ports were drilled along the length of the tailpiece between the P-trap and the strainer and the trap arm between the P-trap and the common line . These holes were fitted with size 00 silicone stoppers Cole-Parmer, Vernon Hills, IL Fig. 5a . Sterile cotton swabs Covidien, Mansfield, MA presoaked in saline were inserted through these sampling ports, and samples were collected by turning the swab in a circular motion on the inner surface ϳ20 cm 2 of the tailpipes. Sample swabs were pulse-vortexed in 3 ml saline, and serial dilutions were plated on TSA. The strainer, faucet aerator, and bowl surface were sampled with presoaked swabs and processed as described earlier. Sink-to-sink transmission of bacteria. To investigate sink-to-sink transmission of bacteria, a distal sink sink 5 Fig. 4 was fitted with a P-trap inoculated with GFP-expressing E. coli. The effects of different inoculum concentrations of GFP-expressing E. coli-10 3 , 10 6 , and Ͼ10 10 CFU/ml colonized for 14 days -were investigated. Identification to the species level of fluorescent and nonfluorescent colonies identified from mixed pipe cultures was performed using a matrix-assisted laser desorption-ionization MALDI -time of flight MALDI-TOF mass spectrometer Vitek-MS; bioMérieux, Durham, NC . The wastewater paths of sinks 1 to 4 were either autoclaved strainer, tailpipe, P-traps, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI . Faucets on each of the five sinks were turned on simultaneously for 1 min, supplying water at a flow rate of 8 liters/min, once every 24 h for 7 days. No additional feed to any of the sinks was added during this period of 7 days. On days 0 and 7, P-traps on each of the five sinks were unfastened, and swab samples from the P-trap were collected and processed as described earlier. Dispersion measured using fluorescent microspheres. Fluoresbrite YO carboxylate microspheres Polysciences, Inc. which had a 1-m diameter and maximum excitation and emission of 529 nm and 546 nm, respectively, were used as a tracer in the preliminary experiments to understand droplet dispersion from the hand-wash sinks. To test whether microspheres could be dispersed from below the sink strainer, a 1-ml suspension of microspheres ϳ10 10 particles was injected through a strainer attached to a Hert 4.5-in. offset drain tailpiece typically used for wheelchair-accessible sinks American Standard, model 7723018.002 Fig. 5c . The vertical distance between the strainer and microsphere suspension injected into the tailpipe was ϳ4 in. Counter space around the sink bowl was thoroughly wiped with alcohol wipes Covidien Webcol 6818; Kendall , and polyester sheets precut to appropriate shapes were placed on the counter to cover the entire sink counter and labeled according to position Fig. 6a . The faucet was turned on for 5 min at a water flow rate of 1.8 to 3.0 liters/min. Polyester sheets were harvested and immediately analyzed using a ChemiDoc MP system Bio-Rad Laboratories, Inc. with an exposure time of 5 s. Fluorescent microspheres were enumerated from the digital micrographs using the Image Lab Software Bio-Rad Laboratories, Inc. . To test whether microspheres could be dispersed from the surface of the sink bowl, the sink bowl was evenly coated with a 20-ml microsphere suspension ϳ10 10 particles/ml using a disposable swab Sage Products, Inc., Cary, IL , and the dispersion experiment was repeated following the protocol described above. To ascertain there was no nonspecific background fluorescence in the sink and/or the water from the faucet, a control using the same protocol but without the fluorescent microspheres was performed before each experiment. Dispersion measured using GFP-expressing E.coli. Dispersion using GFP-expressing E. coli was investigated in three experiments. To test whether live organisms in the P-trap could be dispersed by running water, ϳ10 10 CFU/ml GFP-expressing E. coli in saline was added to an autoclaved P-trap and fitted into the drain line that was preautoclaved strainer, tailpipe, and trap arms . Similarly, to test whether live organisms could be dispersed from the tailpieces of wheelchair-accessible sinks, a suspension of ϳ10 10 CFU/ml GFP-expressing E. coli was added with a syringe through the strainer into the Hert 4.5-ft offset drain tailpiece Fig. 5c . Just as in the microsphere dispersion experiment, the vertical distance between the strainer and GFP-expressing E. coli suspension injected into the tailpipe was ϳ4 in. We next tested whether live organisms from the surface of the sink bowl could be dispersed by running water. The sink bowl surface was evenly coated with an approximately 20-ml suspension of 10 10 CFU/ml GFP-expressing E. coli. Finally, to mimic all of these conditions, a P-trap colonized with GFP-expressing E. coli for 14 days was installed, and a nutrient regimen Fig. 5b was followed for 14 days to intentionally promote the GFP-expressing E. coli colonization in the attached tailpipe and strainer. On day 15, the dispersion experiment was performed. Before each of the GFP-expressing E. coli dispersion experiments, the counter space was thoroughly disinfected with Caviwipes-1. TSA plates were then positioned on the sink counter surrounding the bowl and an extension platform Fig. 6b . Additional plates were attached to the sink bowl, faucets, Plexiglas partitions, and faucet handles using adhesive tape. TSA plates were also placed 3 m away from the sink as negative controls. The faucet was turned on for 5 min with a water flow rate of 1.8 to 3.0 liters/min. Lids of the TSA plates were removed only during faucet operation. Swab samples from the faucet aerators before and after operation were collected and plated on TSA. Prior to each dispersion experiment, 50 ml water from the faucet was also collected, and aliquots were plated to assess for the presence of GFP-expressing E. coli in source water and ensure cross contamination of GFP-expressing E. coli had not occurred. A control dispersion experiment was also performed using the same protocol prior to GFP-expressing E. coli inoculation in each case. Dispersion per defined area CFU per square centimeter was deduced by dividing the CFU counts in the TSA plate by the surface area of the TSA plate.
Why are nucleosides analogs used for chemotheraphy?	they inhibit cellular DNA/RNA polymerases	[ "Nucleoside analogs have been frequently identified as antiviral agents. In recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. Nucleoside analogs generally interfere with cellular nucleos t ide synthesis pathways, resulting in the depletion or imbalance of d NTP pools. Intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos t ide synthesis inhibition. The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos t ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs.", "The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos t ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs. Text: Nucleoside analogs have been historically used for anti-cancer chemotherapy because they inhibit cellular DNA/RNA polymerases . More recently, nucleoside analogs have expanded their therapeutic applications and are being used to develop antiviral drugs against a wide range of serious and life-threatening viruses. Some nucleoside analog drugs targeting specific viral polymerases acyclovir for herpesviruses, zidovudine for human immunodeficiency virus HIV , and sofosbuvir for hepatitis C virus HCV have been successful in clinical trials and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α .", "Some nucleoside analog drugs targeting specific viral polymerases acyclovir for herpesviruses, zidovudine for human immunodeficiency virus HIV , and sofosbuvir for hepatitis C virus HCV have been successful in clinical trials and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α . Importantly, extensive studies on the antiviral action of ribavirin have established the underlying molecular framework of nucleoside analogs. The primary mechanism to explain the antiviral effect of nucleoside analogs is based on their direct action on viral polymerization. Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 .", "Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools.", "There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferon-stimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated.", "Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors.", "In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways .", "The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity.", "A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group.", "They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers .", "Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 .", "MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV .", "Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV EC 50 of 1.2 µM and 4.9 µM, respectively .", "To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV EC 50 of 1.2 µM and 4.9 µM, respectively . More recently, gemcitabine was shown to effectively suppress ZIKV infection and replication in human retinal pigment epithelium RPE cells, particularly at non-cytotoxic concentrations EC 50 of 0.01 µM vs. CC 50 of > 10 µM . ZIKV, a member of the Flaviviridae family, can infect pregnant women and cause congenital abnormalities such as microcephaly in infants, which has attracted increasing public attention as well as extensive research and development into possible treatments. Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively , which were lower concentrations than those used in cancer therapy . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency .", "Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively , which were lower concentrations than those used in cancer therapy . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency . In a follow up study, Clouser et al. further reported the antiviral effect of gemcitabine against HIV-related retrovirus, murine leukemia virus MuLV , in vitro EC 50 of 1.6 nM and even in murine AIDS model . A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. EC 50 of 0.068 µM .", "A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. EC 50 of 0.068 µM . They also tested whether gemcitabine had an antiviral effect on several other viruses of different families and found its strong inhibitory effect on Sindbis virus and herpes simplex virus-1 HSV-1 >2 log reduction in virus titer but relatively weak effects on Semliki forest virus and human echovirus 6, and minimal effects on Bunyamwera virus, measles virus MeV , and vaccinia virus . The antiviral effect of gemcitabine on EVs, initially performed on Coxsackievirus B3 CVB3 , was found from screening FDA-approved drugs in CVB3 replicon-harboring Vero cells by our group EC 50 of 0.4 µM . Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 EV71 and human rhinoviruses HRVs EC 50 s of 1 and 1-5 µM, respectively . In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model .", "Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 EV71 and human rhinoviruses HRVs EC 50 s of 1 and 1-5 µM, respectively . In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model . In this study, intranasal administration of gemcitabine significantly lowered the pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including TNF-α and IL-1β, and the number of lung infiltrating lymphocytes. More recently, Zhang et al. also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells EC 50 of 0.3 µM . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases.", "also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells EC 50 of 0.3 µM . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases. Moreover, it is possible that gemcitabine is effective for other untested RNA viruses. Because gemcitabine is a deoxycytidine analog that interferes with DNA as well as RNA synthesis, DNA viruses may not be the exception. Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments.", "Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments. This is especially true for newly emerging or re-emerged viruses involving serious illnesses, such as MERS-CoV, SARS-CoV, and ZIKV, which are major threats to public health and which urgently need an effective treatment during their early stages of infection. In this regard, repurposing of gemcitabine for the treatment of patients infected with these deadly viruses is a realistic approach. Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents.", "Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents. In this manner, an effective antiviral treatment may be achieved by the synergistic action of two antivirals with much lower doses for each drug, which minimizes deleterious side effects when used clinically. As an example, the synergistic antiviral effect of gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few RNA viruses, was reported against EVs such as CVB3 and EV71 . As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials.", "As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials. Even though most antiviral data have originated from in vitro studies, two recent studies have reported the antiviral effects of gemcitabine in murine models . More extensive analyses of gemcitabine in animal models in the near future will accelerate its therapeutic applications in clinical trials. Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis .", "Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis . Moreover, gemcitabine strongly induced the expression of several ISGs including CXCL10, IRF7, IRF9, IFIT1, and DDX58, which were the major effectors in the innate immunity that defended the host against the virus infection. These results were consistent with a previous report that gemcitabine stimulated the production of IFN-β and IFN-γ in IAV-infected RPE cells . Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 6, 10, .", "Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 6, 10, . Regarding purine biosynthesis inhibitors, ribavirin and mycophenolic acid MPA are inhibitors of inosine-5 -monophosphate IMP dehydrogenase IMPDH , which is a key enzyme of the purine biosynthesis pathway. These inhibitors have been successfully used as clinical antiviral or immunosuppressant agents for decades. Both have antiviral activities against viruses such as HCV, hepatitis E virus HEV , MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus WNV , Chikungunya virus CHIKV , and IAV , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses .", "Both have antiviral activities against viruses such as HCV, hepatitis E virus HEV , MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus WNV , Chikungunya virus CHIKV , and IAV , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses . ISG activation occurred through an undefined mechanism that was different from the classical IFN signaling, intracellular dsRNA sensing pathway, Toll-like receptor and nuclear factor B pathways. More importantly, ribavirin-induced ISG activation and antiviral activity were suppressed using supplemented guanosine, a natural analog of ribavirin, suggesting IMPDH inhibition-mediated ISG activation as an alternative innate immunity pathway. Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin .", "Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin . Similar results were obtained with several IMPDH1 or IMPDH2 inhibitors, with various affinities, that were custom-designed and synthesized . As shown in Table 2 , most pyrimidine biosynthesis inhibitors target dihydroorotate dehydrogenase DHODH , an essential enzyme in de novo pyrimidine synthesis. Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element ISRE -stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV .", "Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element ISRE -stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV . DD264 enhanced the expression of several ISGs, which were almost completely suppressed by the addition of supplemented uridine, indicating DHODH inhibition-mediated ISG activation. Moreover, the antiviral activity of and ISG activation by DD264 required the interferon regulatory factor 1 IRF1 transcription factor, a master regulator of antiviral gene expression , which was consistent with the observation that the anti-HCV activity of MPA was partially mediated by IRF1 . In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 .", "In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 . However, whether ISG activation is mediated by pyrimidine biosynthesis inhibition remains to be determined. The mechanism of nucleotide synthesis inhibitor-induced ISG activation is still presently unclear. Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal . First, Wang et al.", "Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal . First, Wang et al. clearly showed that ISG activation and anti-HEV activity induced by MPA or brequinar was not mediated by JAK . Second, IRF7 induction by ribavirin was not affected by knockdown of STAT1, while that of IFN-α was strongly affected under the same conditions . Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine .", "Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine . Moreover, the upregulation of DDX58 mRNAs induced by gemcitabine was not affected by IRF9 knockdown, which was contrary to the result that IFN-α-induced upregulation of DDX58 mRNAs was significantly suppressed under the same conditions. Consistent with above observations, there have been some reports that ISGs was induced in the absence of JAK1 or STAT1 activation . Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the d NTP pool.", "Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the d NTP pool. Inactivation of metabolic enzyme s itself or consequently altered nucleos t ide pools might trigger a signal, which is ultimately delivered to certain cis-acting elements on the promoter of a subset of ISGs, possibly through the relay of kinases and transcription factors. Based on the previously mentioned reports, this signal is less likely to be dependent on STAT1/2-IRF9 IFN-stimulated gene factor 3; ISGF3 , at least for gemcitabine, which is the major transcriptional complex in the IFN-induced JAK/STAT pathway. It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin .", "It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin . Despite the consensus of ISG activation, each purine/pyrimidine biosynthesis inhibitor seems to induce distinct sets of ISGs, at least with different patterns . Targeting an enzyme in which pathways purine or pyrimidine synthesis or steps early/late and de novo/salvage produce different levels of intermediates and nucleos t ides will consequently result in diverse outcomes of ISG activations. There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation.", "There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation. Systematic analyses of signaling kinases, IRFs, and STATs using siRNA knockdown and/or pharmacological inhibition and metabolic analyses of corresponding intermediates and nucleos t ides should therefore clarify the underlying molecular mechanisms of ISG activation by purine/pyrimidine biosynthesis inhibitors. As newly emerging or re-emerged viruses such as SARS-CoV, MERS-CoV, and ZIKV have become a major threat to public health, the need for broad-spectrum antiviral drug has increased. In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs.", "In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs. Nucleoside analogs probably induce different subsets of ISGs, at least with a different pattern, leading to various combinations of ISGs and resulting antiviral outcomes. Moreover, according to Schoggins et al., different viruses are affected by distinct subsets of ISGs and some ISGs such as IRF1, MB21D1, HPSE, DDX58, MDA, and IFITM3 act broadly on various viruses . Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives.", "Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives. Nevertheless, nucleoside analogs interfering with the host nucleotide synthesis pathway suggest possible side effects in their clinical applications. Careful evaluation of clinical safety is required and their application for the urgent measure of patients infected with deadly viruses would be worth being primarily considered." ]	1,594	5,232	Nucleoside analogs have been frequently identified as antiviral agents. In recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. Nucleoside analogs generally interfere with cellular nucleos t ide synthesis pathways, resulting in the depletion or imbalance of d NTP pools. Intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos t ide synthesis inhibition. The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos t ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs. Text: Nucleoside analogs have been historically used for anti-cancer chemotherapy because they inhibit cellular DNA/RNA polymerases . More recently, nucleoside analogs have expanded their therapeutic applications and are being used to develop antiviral drugs against a wide range of serious and life-threatening viruses. Some nucleoside analog drugs targeting specific viral polymerases acyclovir for herpesviruses, zidovudine for human immunodeficiency virus HIV , and sofosbuvir for hepatitis C virus HCV have been successful in clinical trials and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α . Importantly, extensive studies on the antiviral action of ribavirin have established the underlying molecular framework of nucleoside analogs. The primary mechanism to explain the antiviral effect of nucleoside analogs is based on their direct action on viral polymerization. Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferon-stimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV EC 50 of 1.2 µM and 4.9 µM, respectively . More recently, gemcitabine was shown to effectively suppress ZIKV infection and replication in human retinal pigment epithelium RPE cells, particularly at non-cytotoxic concentrations EC 50 of 0.01 µM vs. CC 50 of > 10 µM . ZIKV, a member of the Flaviviridae family, can infect pregnant women and cause congenital abnormalities such as microcephaly in infants, which has attracted increasing public attention as well as extensive research and development into possible treatments. Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively , which were lower concentrations than those used in cancer therapy . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency . In a follow up study, Clouser et al. further reported the antiviral effect of gemcitabine against HIV-related retrovirus, murine leukemia virus MuLV , in vitro EC 50 of 1.6 nM and even in murine AIDS model . A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. EC 50 of 0.068 µM . They also tested whether gemcitabine had an antiviral effect on several other viruses of different families and found its strong inhibitory effect on Sindbis virus and herpes simplex virus-1 HSV-1 >2 log reduction in virus titer but relatively weak effects on Semliki forest virus and human echovirus 6, and minimal effects on Bunyamwera virus, measles virus MeV , and vaccinia virus . The antiviral effect of gemcitabine on EVs, initially performed on Coxsackievirus B3 CVB3 , was found from screening FDA-approved drugs in CVB3 replicon-harboring Vero cells by our group EC 50 of 0.4 µM . Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 EV71 and human rhinoviruses HRVs EC 50 s of 1 and 1-5 µM, respectively . In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model . In this study, intranasal administration of gemcitabine significantly lowered the pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including TNF-α and IL-1β, and the number of lung infiltrating lymphocytes. More recently, Zhang et al. also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells EC 50 of 0.3 µM . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases. Moreover, it is possible that gemcitabine is effective for other untested RNA viruses. Because gemcitabine is a deoxycytidine analog that interferes with DNA as well as RNA synthesis, DNA viruses may not be the exception. Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments. This is especially true for newly emerging or re-emerged viruses involving serious illnesses, such as MERS-CoV, SARS-CoV, and ZIKV, which are major threats to public health and which urgently need an effective treatment during their early stages of infection. In this regard, repurposing of gemcitabine for the treatment of patients infected with these deadly viruses is a realistic approach. Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents. In this manner, an effective antiviral treatment may be achieved by the synergistic action of two antivirals with much lower doses for each drug, which minimizes deleterious side effects when used clinically. As an example, the synergistic antiviral effect of gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few RNA viruses, was reported against EVs such as CVB3 and EV71 . As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials. Even though most antiviral data have originated from in vitro studies, two recent studies have reported the antiviral effects of gemcitabine in murine models . More extensive analyses of gemcitabine in animal models in the near future will accelerate its therapeutic applications in clinical trials. Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis . Moreover, gemcitabine strongly induced the expression of several ISGs including CXCL10, IRF7, IRF9, IFIT1, and DDX58, which were the major effectors in the innate immunity that defended the host against the virus infection. These results were consistent with a previous report that gemcitabine stimulated the production of IFN-β and IFN-γ in IAV-infected RPE cells . Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 6, 10, . Regarding purine biosynthesis inhibitors, ribavirin and mycophenolic acid MPA are inhibitors of inosine-5 -monophosphate IMP dehydrogenase IMPDH , which is a key enzyme of the purine biosynthesis pathway. These inhibitors have been successfully used as clinical antiviral or immunosuppressant agents for decades. Both have antiviral activities against viruses such as HCV, hepatitis E virus HEV , MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus WNV , Chikungunya virus CHIKV , and IAV , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses . ISG activation occurred through an undefined mechanism that was different from the classical IFN signaling, intracellular dsRNA sensing pathway, Toll-like receptor and nuclear factor B pathways. More importantly, ribavirin-induced ISG activation and antiviral activity were suppressed using supplemented guanosine, a natural analog of ribavirin, suggesting IMPDH inhibition-mediated ISG activation as an alternative innate immunity pathway. Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin . Similar results were obtained with several IMPDH1 or IMPDH2 inhibitors, with various affinities, that were custom-designed and synthesized . As shown in Table 2 , most pyrimidine biosynthesis inhibitors target dihydroorotate dehydrogenase DHODH , an essential enzyme in de novo pyrimidine synthesis. Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element ISRE -stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV . DD264 enhanced the expression of several ISGs, which were almost completely suppressed by the addition of supplemented uridine, indicating DHODH inhibition-mediated ISG activation. Moreover, the antiviral activity of and ISG activation by DD264 required the interferon regulatory factor 1 IRF1 transcription factor, a master regulator of antiviral gene expression , which was consistent with the observation that the anti-HCV activity of MPA was partially mediated by IRF1 . In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 . However, whether ISG activation is mediated by pyrimidine biosynthesis inhibition remains to be determined. The mechanism of nucleotide synthesis inhibitor-induced ISG activation is still presently unclear. Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal . First, Wang et al. clearly showed that ISG activation and anti-HEV activity induced by MPA or brequinar was not mediated by JAK . Second, IRF7 induction by ribavirin was not affected by knockdown of STAT1, while that of IFN-α was strongly affected under the same conditions . Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine . Moreover, the upregulation of DDX58 mRNAs induced by gemcitabine was not affected by IRF9 knockdown, which was contrary to the result that IFN-α-induced upregulation of DDX58 mRNAs was significantly suppressed under the same conditions. Consistent with above observations, there have been some reports that ISGs was induced in the absence of JAK1 or STAT1 activation . Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the d NTP pool. Inactivation of metabolic enzyme s itself or consequently altered nucleos t ide pools might trigger a signal, which is ultimately delivered to certain cis-acting elements on the promoter of a subset of ISGs, possibly through the relay of kinases and transcription factors. Based on the previously mentioned reports, this signal is less likely to be dependent on STAT1/2-IRF9 IFN-stimulated gene factor 3; ISGF3 , at least for gemcitabine, which is the major transcriptional complex in the IFN-induced JAK/STAT pathway. It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin . Despite the consensus of ISG activation, each purine/pyrimidine biosynthesis inhibitor seems to induce distinct sets of ISGs, at least with different patterns . Targeting an enzyme in which pathways purine or pyrimidine synthesis or steps early/late and de novo/salvage produce different levels of intermediates and nucleos t ides will consequently result in diverse outcomes of ISG activations. There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation. Systematic analyses of signaling kinases, IRFs, and STATs using siRNA knockdown and/or pharmacological inhibition and metabolic analyses of corresponding intermediates and nucleos t ides should therefore clarify the underlying molecular mechanisms of ISG activation by purine/pyrimidine biosynthesis inhibitors. As newly emerging or re-emerged viruses such as SARS-CoV, MERS-CoV, and ZIKV have become a major threat to public health, the need for broad-spectrum antiviral drug has increased. In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs. Nucleoside analogs probably induce different subsets of ISGs, at least with a different pattern, leading to various combinations of ISGs and resulting antiviral outcomes. Moreover, according to Schoggins et al., different viruses are affected by distinct subsets of ISGs and some ISGs such as IRF1, MB21D1, HPSE, DDX58, MDA, and IFITM3 act broadly on various viruses . Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives. Nevertheless, nucleoside analogs interfering with the host nucleotide synthesis pathway suggest possible side effects in their clinical applications. Careful evaluation of clinical safety is required and their application for the urgent measure of patients infected with deadly viruses would be worth being primarily considered.
What nucleoside analog is the focus of the current study?	gemcitabine	[ "Nucleoside analogs have been frequently identified as antiviral agents. In recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. Nucleoside analogs generally interfere with cellular nucleos t ide synthesis pathways, resulting in the depletion or imbalance of d NTP pools. Intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos t ide synthesis inhibition. The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos t ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs.", "The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos t ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs. Text: Nucleoside analogs have been historically used for anti-cancer chemotherapy because they inhibit cellular DNA/RNA polymerases . More recently, nucleoside analogs have expanded their therapeutic applications and are being used to develop antiviral drugs against a wide range of serious and life-threatening viruses. Some nucleoside analog drugs targeting specific viral polymerases acyclovir for herpesviruses, zidovudine for human immunodeficiency virus HIV , and sofosbuvir for hepatitis C virus HCV have been successful in clinical trials and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α .", "Some nucleoside analog drugs targeting specific viral polymerases acyclovir for herpesviruses, zidovudine for human immunodeficiency virus HIV , and sofosbuvir for hepatitis C virus HCV have been successful in clinical trials and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α . Importantly, extensive studies on the antiviral action of ribavirin have established the underlying molecular framework of nucleoside analogs. The primary mechanism to explain the antiviral effect of nucleoside analogs is based on their direct action on viral polymerization. Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 .", "Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools.", "There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferon-stimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated.", "Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors.", "In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways .", "The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity.", "A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group.", "They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers .", "Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 .", "MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV .", "Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV EC 50 of 1.2 µM and 4.9 µM, respectively .", "To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV EC 50 of 1.2 µM and 4.9 µM, respectively . More recently, gemcitabine was shown to effectively suppress ZIKV infection and replication in human retinal pigment epithelium RPE cells, particularly at non-cytotoxic concentrations EC 50 of 0.01 µM vs. CC 50 of > 10 µM . ZIKV, a member of the Flaviviridae family, can infect pregnant women and cause congenital abnormalities such as microcephaly in infants, which has attracted increasing public attention as well as extensive research and development into possible treatments. Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively , which were lower concentrations than those used in cancer therapy . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency .", "Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively , which were lower concentrations than those used in cancer therapy . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency . In a follow up study, Clouser et al. further reported the antiviral effect of gemcitabine against HIV-related retrovirus, murine leukemia virus MuLV , in vitro EC 50 of 1.6 nM and even in murine AIDS model . A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. EC 50 of 0.068 µM .", "A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. EC 50 of 0.068 µM . They also tested whether gemcitabine had an antiviral effect on several other viruses of different families and found its strong inhibitory effect on Sindbis virus and herpes simplex virus-1 HSV-1 >2 log reduction in virus titer but relatively weak effects on Semliki forest virus and human echovirus 6, and minimal effects on Bunyamwera virus, measles virus MeV , and vaccinia virus . The antiviral effect of gemcitabine on EVs, initially performed on Coxsackievirus B3 CVB3 , was found from screening FDA-approved drugs in CVB3 replicon-harboring Vero cells by our group EC 50 of 0.4 µM . Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 EV71 and human rhinoviruses HRVs EC 50 s of 1 and 1-5 µM, respectively . In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model .", "Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 EV71 and human rhinoviruses HRVs EC 50 s of 1 and 1-5 µM, respectively . In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model . In this study, intranasal administration of gemcitabine significantly lowered the pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including TNF-α and IL-1β, and the number of lung infiltrating lymphocytes. More recently, Zhang et al. also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells EC 50 of 0.3 µM . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases.", "also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells EC 50 of 0.3 µM . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases. Moreover, it is possible that gemcitabine is effective for other untested RNA viruses. Because gemcitabine is a deoxycytidine analog that interferes with DNA as well as RNA synthesis, DNA viruses may not be the exception. Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments.", "Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments. This is especially true for newly emerging or re-emerged viruses involving serious illnesses, such as MERS-CoV, SARS-CoV, and ZIKV, which are major threats to public health and which urgently need an effective treatment during their early stages of infection. In this regard, repurposing of gemcitabine for the treatment of patients infected with these deadly viruses is a realistic approach. Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents.", "Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents. In this manner, an effective antiviral treatment may be achieved by the synergistic action of two antivirals with much lower doses for each drug, which minimizes deleterious side effects when used clinically. As an example, the synergistic antiviral effect of gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few RNA viruses, was reported against EVs such as CVB3 and EV71 . As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials.", "As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials. Even though most antiviral data have originated from in vitro studies, two recent studies have reported the antiviral effects of gemcitabine in murine models . More extensive analyses of gemcitabine in animal models in the near future will accelerate its therapeutic applications in clinical trials. Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis .", "Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis . Moreover, gemcitabine strongly induced the expression of several ISGs including CXCL10, IRF7, IRF9, IFIT1, and DDX58, which were the major effectors in the innate immunity that defended the host against the virus infection. These results were consistent with a previous report that gemcitabine stimulated the production of IFN-β and IFN-γ in IAV-infected RPE cells . Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 6, 10, .", "Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 6, 10, . Regarding purine biosynthesis inhibitors, ribavirin and mycophenolic acid MPA are inhibitors of inosine-5 -monophosphate IMP dehydrogenase IMPDH , which is a key enzyme of the purine biosynthesis pathway. These inhibitors have been successfully used as clinical antiviral or immunosuppressant agents for decades. Both have antiviral activities against viruses such as HCV, hepatitis E virus HEV , MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus WNV , Chikungunya virus CHIKV , and IAV , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses .", "Both have antiviral activities against viruses such as HCV, hepatitis E virus HEV , MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus WNV , Chikungunya virus CHIKV , and IAV , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses . ISG activation occurred through an undefined mechanism that was different from the classical IFN signaling, intracellular dsRNA sensing pathway, Toll-like receptor and nuclear factor B pathways. More importantly, ribavirin-induced ISG activation and antiviral activity were suppressed using supplemented guanosine, a natural analog of ribavirin, suggesting IMPDH inhibition-mediated ISG activation as an alternative innate immunity pathway. Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin .", "Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin . Similar results were obtained with several IMPDH1 or IMPDH2 inhibitors, with various affinities, that were custom-designed and synthesized . As shown in Table 2 , most pyrimidine biosynthesis inhibitors target dihydroorotate dehydrogenase DHODH , an essential enzyme in de novo pyrimidine synthesis. Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element ISRE -stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV .", "Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element ISRE -stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV . DD264 enhanced the expression of several ISGs, which were almost completely suppressed by the addition of supplemented uridine, indicating DHODH inhibition-mediated ISG activation. Moreover, the antiviral activity of and ISG activation by DD264 required the interferon regulatory factor 1 IRF1 transcription factor, a master regulator of antiviral gene expression , which was consistent with the observation that the anti-HCV activity of MPA was partially mediated by IRF1 . In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 .", "In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 . However, whether ISG activation is mediated by pyrimidine biosynthesis inhibition remains to be determined. The mechanism of nucleotide synthesis inhibitor-induced ISG activation is still presently unclear. Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal . First, Wang et al.", "Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal . First, Wang et al. clearly showed that ISG activation and anti-HEV activity induced by MPA or brequinar was not mediated by JAK . Second, IRF7 induction by ribavirin was not affected by knockdown of STAT1, while that of IFN-α was strongly affected under the same conditions . Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine .", "Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine . Moreover, the upregulation of DDX58 mRNAs induced by gemcitabine was not affected by IRF9 knockdown, which was contrary to the result that IFN-α-induced upregulation of DDX58 mRNAs was significantly suppressed under the same conditions. Consistent with above observations, there have been some reports that ISGs was induced in the absence of JAK1 or STAT1 activation . Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the d NTP pool.", "Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the d NTP pool. Inactivation of metabolic enzyme s itself or consequently altered nucleos t ide pools might trigger a signal, which is ultimately delivered to certain cis-acting elements on the promoter of a subset of ISGs, possibly through the relay of kinases and transcription factors. Based on the previously mentioned reports, this signal is less likely to be dependent on STAT1/2-IRF9 IFN-stimulated gene factor 3; ISGF3 , at least for gemcitabine, which is the major transcriptional complex in the IFN-induced JAK/STAT pathway. It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin .", "It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin . Despite the consensus of ISG activation, each purine/pyrimidine biosynthesis inhibitor seems to induce distinct sets of ISGs, at least with different patterns . Targeting an enzyme in which pathways purine or pyrimidine synthesis or steps early/late and de novo/salvage produce different levels of intermediates and nucleos t ides will consequently result in diverse outcomes of ISG activations. There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation.", "There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation. Systematic analyses of signaling kinases, IRFs, and STATs using siRNA knockdown and/or pharmacological inhibition and metabolic analyses of corresponding intermediates and nucleos t ides should therefore clarify the underlying molecular mechanisms of ISG activation by purine/pyrimidine biosynthesis inhibitors. As newly emerging or re-emerged viruses such as SARS-CoV, MERS-CoV, and ZIKV have become a major threat to public health, the need for broad-spectrum antiviral drug has increased. In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs.", "In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs. Nucleoside analogs probably induce different subsets of ISGs, at least with a different pattern, leading to various combinations of ISGs and resulting antiviral outcomes. Moreover, according to Schoggins et al., different viruses are affected by distinct subsets of ISGs and some ISGs such as IRF1, MB21D1, HPSE, DDX58, MDA, and IFITM3 act broadly on various viruses . Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives.", "Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives. Nevertheless, nucleoside analogs interfering with the host nucleotide synthesis pathway suggest possible side effects in their clinical applications. Careful evaluation of clinical safety is required and their application for the urgent measure of patients infected with deadly viruses would be worth being primarily considered." ]	1,594	5,233	Nucleoside analogs have been frequently identified as antiviral agents. In recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. Nucleoside analogs generally interfere with cellular nucleos t ide synthesis pathways, resulting in the depletion or imbalance of d NTP pools. Intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos t ide synthesis inhibition. The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos t ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs. Text: Nucleoside analogs have been historically used for anti-cancer chemotherapy because they inhibit cellular DNA/RNA polymerases . More recently, nucleoside analogs have expanded their therapeutic applications and are being used to develop antiviral drugs against a wide range of serious and life-threatening viruses. Some nucleoside analog drugs targeting specific viral polymerases acyclovir for herpesviruses, zidovudine for human immunodeficiency virus HIV , and sofosbuvir for hepatitis C virus HCV have been successful in clinical trials and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α . Importantly, extensive studies on the antiviral action of ribavirin have established the underlying molecular framework of nucleoside analogs. The primary mechanism to explain the antiviral effect of nucleoside analogs is based on their direct action on viral polymerization. Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferon-stimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV EC 50 of 1.2 µM and 4.9 µM, respectively . More recently, gemcitabine was shown to effectively suppress ZIKV infection and replication in human retinal pigment epithelium RPE cells, particularly at non-cytotoxic concentrations EC 50 of 0.01 µM vs. CC 50 of > 10 µM . ZIKV, a member of the Flaviviridae family, can infect pregnant women and cause congenital abnormalities such as microcephaly in infants, which has attracted increasing public attention as well as extensive research and development into possible treatments. Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively , which were lower concentrations than those used in cancer therapy . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency . In a follow up study, Clouser et al. further reported the antiviral effect of gemcitabine against HIV-related retrovirus, murine leukemia virus MuLV , in vitro EC 50 of 1.6 nM and even in murine AIDS model . A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. EC 50 of 0.068 µM . They also tested whether gemcitabine had an antiviral effect on several other viruses of different families and found its strong inhibitory effect on Sindbis virus and herpes simplex virus-1 HSV-1 >2 log reduction in virus titer but relatively weak effects on Semliki forest virus and human echovirus 6, and minimal effects on Bunyamwera virus, measles virus MeV , and vaccinia virus . The antiviral effect of gemcitabine on EVs, initially performed on Coxsackievirus B3 CVB3 , was found from screening FDA-approved drugs in CVB3 replicon-harboring Vero cells by our group EC 50 of 0.4 µM . Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 EV71 and human rhinoviruses HRVs EC 50 s of 1 and 1-5 µM, respectively . In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model . In this study, intranasal administration of gemcitabine significantly lowered the pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including TNF-α and IL-1β, and the number of lung infiltrating lymphocytes. More recently, Zhang et al. also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells EC 50 of 0.3 µM . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases. Moreover, it is possible that gemcitabine is effective for other untested RNA viruses. Because gemcitabine is a deoxycytidine analog that interferes with DNA as well as RNA synthesis, DNA viruses may not be the exception. Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments. This is especially true for newly emerging or re-emerged viruses involving serious illnesses, such as MERS-CoV, SARS-CoV, and ZIKV, which are major threats to public health and which urgently need an effective treatment during their early stages of infection. In this regard, repurposing of gemcitabine for the treatment of patients infected with these deadly viruses is a realistic approach. Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents. In this manner, an effective antiviral treatment may be achieved by the synergistic action of two antivirals with much lower doses for each drug, which minimizes deleterious side effects when used clinically. As an example, the synergistic antiviral effect of gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few RNA viruses, was reported against EVs such as CVB3 and EV71 . As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials. Even though most antiviral data have originated from in vitro studies, two recent studies have reported the antiviral effects of gemcitabine in murine models . More extensive analyses of gemcitabine in animal models in the near future will accelerate its therapeutic applications in clinical trials. Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis . Moreover, gemcitabine strongly induced the expression of several ISGs including CXCL10, IRF7, IRF9, IFIT1, and DDX58, which were the major effectors in the innate immunity that defended the host against the virus infection. These results were consistent with a previous report that gemcitabine stimulated the production of IFN-β and IFN-γ in IAV-infected RPE cells . Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 6, 10, . Regarding purine biosynthesis inhibitors, ribavirin and mycophenolic acid MPA are inhibitors of inosine-5 -monophosphate IMP dehydrogenase IMPDH , which is a key enzyme of the purine biosynthesis pathway. These inhibitors have been successfully used as clinical antiviral or immunosuppressant agents for decades. Both have antiviral activities against viruses such as HCV, hepatitis E virus HEV , MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus WNV , Chikungunya virus CHIKV , and IAV , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses . ISG activation occurred through an undefined mechanism that was different from the classical IFN signaling, intracellular dsRNA sensing pathway, Toll-like receptor and nuclear factor B pathways. More importantly, ribavirin-induced ISG activation and antiviral activity were suppressed using supplemented guanosine, a natural analog of ribavirin, suggesting IMPDH inhibition-mediated ISG activation as an alternative innate immunity pathway. Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin . Similar results were obtained with several IMPDH1 or IMPDH2 inhibitors, with various affinities, that were custom-designed and synthesized . As shown in Table 2 , most pyrimidine biosynthesis inhibitors target dihydroorotate dehydrogenase DHODH , an essential enzyme in de novo pyrimidine synthesis. Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element ISRE -stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV . DD264 enhanced the expression of several ISGs, which were almost completely suppressed by the addition of supplemented uridine, indicating DHODH inhibition-mediated ISG activation. Moreover, the antiviral activity of and ISG activation by DD264 required the interferon regulatory factor 1 IRF1 transcription factor, a master regulator of antiviral gene expression , which was consistent with the observation that the anti-HCV activity of MPA was partially mediated by IRF1 . In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 . However, whether ISG activation is mediated by pyrimidine biosynthesis inhibition remains to be determined. The mechanism of nucleotide synthesis inhibitor-induced ISG activation is still presently unclear. Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal . First, Wang et al. clearly showed that ISG activation and anti-HEV activity induced by MPA or brequinar was not mediated by JAK . Second, IRF7 induction by ribavirin was not affected by knockdown of STAT1, while that of IFN-α was strongly affected under the same conditions . Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine . Moreover, the upregulation of DDX58 mRNAs induced by gemcitabine was not affected by IRF9 knockdown, which was contrary to the result that IFN-α-induced upregulation of DDX58 mRNAs was significantly suppressed under the same conditions. Consistent with above observations, there have been some reports that ISGs was induced in the absence of JAK1 or STAT1 activation . Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the d NTP pool. Inactivation of metabolic enzyme s itself or consequently altered nucleos t ide pools might trigger a signal, which is ultimately delivered to certain cis-acting elements on the promoter of a subset of ISGs, possibly through the relay of kinases and transcription factors. Based on the previously mentioned reports, this signal is less likely to be dependent on STAT1/2-IRF9 IFN-stimulated gene factor 3; ISGF3 , at least for gemcitabine, which is the major transcriptional complex in the IFN-induced JAK/STAT pathway. It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin . Despite the consensus of ISG activation, each purine/pyrimidine biosynthesis inhibitor seems to induce distinct sets of ISGs, at least with different patterns . Targeting an enzyme in which pathways purine or pyrimidine synthesis or steps early/late and de novo/salvage produce different levels of intermediates and nucleos t ides will consequently result in diverse outcomes of ISG activations. There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation. Systematic analyses of signaling kinases, IRFs, and STATs using siRNA knockdown and/or pharmacological inhibition and metabolic analyses of corresponding intermediates and nucleos t ides should therefore clarify the underlying molecular mechanisms of ISG activation by purine/pyrimidine biosynthesis inhibitors. As newly emerging or re-emerged viruses such as SARS-CoV, MERS-CoV, and ZIKV have become a major threat to public health, the need for broad-spectrum antiviral drug has increased. In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs. Nucleoside analogs probably induce different subsets of ISGs, at least with a different pattern, leading to various combinations of ISGs and resulting antiviral outcomes. Moreover, according to Schoggins et al., different viruses are affected by distinct subsets of ISGs and some ISGs such as IRF1, MB21D1, HPSE, DDX58, MDA, and IFITM3 act broadly on various viruses . Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives. Nevertheless, nucleoside analogs interfering with the host nucleotide synthesis pathway suggest possible side effects in their clinical applications. Careful evaluation of clinical safety is required and their application for the urgent measure of patients infected with deadly viruses would be worth being primarily considered.
Gemcitabine has been shown to have antiviral activity against which viruses?	Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), Zika virus (ZIKV), HCV, poliovirus (PV), influenza A virus (IAV), HIV, and enteroviruses (EV)	[ "Nucleoside analogs have been frequently identified as antiviral agents. In recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. Nucleoside analogs generally interfere with cellular nucleos t ide synthesis pathways, resulting in the depletion or imbalance of d NTP pools. Intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos t ide synthesis inhibition. The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos t ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs.", "The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos t ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs. Text: Nucleoside analogs have been historically used for anti-cancer chemotherapy because they inhibit cellular DNA/RNA polymerases . More recently, nucleoside analogs have expanded their therapeutic applications and are being used to develop antiviral drugs against a wide range of serious and life-threatening viruses. Some nucleoside analog drugs targeting specific viral polymerases acyclovir for herpesviruses, zidovudine for human immunodeficiency virus HIV , and sofosbuvir for hepatitis C virus HCV have been successful in clinical trials and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α .", "Some nucleoside analog drugs targeting specific viral polymerases acyclovir for herpesviruses, zidovudine for human immunodeficiency virus HIV , and sofosbuvir for hepatitis C virus HCV have been successful in clinical trials and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α . Importantly, extensive studies on the antiviral action of ribavirin have established the underlying molecular framework of nucleoside analogs. The primary mechanism to explain the antiviral effect of nucleoside analogs is based on their direct action on viral polymerization. Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 .", "Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools.", "There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferon-stimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated.", "Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors.", "In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways .", "The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity.", "A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group.", "They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers .", "Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 .", "MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV .", "Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV EC 50 of 1.2 µM and 4.9 µM, respectively .", "To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV EC 50 of 1.2 µM and 4.9 µM, respectively . More recently, gemcitabine was shown to effectively suppress ZIKV infection and replication in human retinal pigment epithelium RPE cells, particularly at non-cytotoxic concentrations EC 50 of 0.01 µM vs. CC 50 of > 10 µM . ZIKV, a member of the Flaviviridae family, can infect pregnant women and cause congenital abnormalities such as microcephaly in infants, which has attracted increasing public attention as well as extensive research and development into possible treatments. Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively , which were lower concentrations than those used in cancer therapy . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency .", "Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively , which were lower concentrations than those used in cancer therapy . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency . In a follow up study, Clouser et al. further reported the antiviral effect of gemcitabine against HIV-related retrovirus, murine leukemia virus MuLV , in vitro EC 50 of 1.6 nM and even in murine AIDS model . A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. EC 50 of 0.068 µM .", "A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. EC 50 of 0.068 µM . They also tested whether gemcitabine had an antiviral effect on several other viruses of different families and found its strong inhibitory effect on Sindbis virus and herpes simplex virus-1 HSV-1 >2 log reduction in virus titer but relatively weak effects on Semliki forest virus and human echovirus 6, and minimal effects on Bunyamwera virus, measles virus MeV , and vaccinia virus . The antiviral effect of gemcitabine on EVs, initially performed on Coxsackievirus B3 CVB3 , was found from screening FDA-approved drugs in CVB3 replicon-harboring Vero cells by our group EC 50 of 0.4 µM . Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 EV71 and human rhinoviruses HRVs EC 50 s of 1 and 1-5 µM, respectively . In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model .", "Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 EV71 and human rhinoviruses HRVs EC 50 s of 1 and 1-5 µM, respectively . In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model . In this study, intranasal administration of gemcitabine significantly lowered the pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including TNF-α and IL-1β, and the number of lung infiltrating lymphocytes. More recently, Zhang et al. also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells EC 50 of 0.3 µM . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases.", "also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells EC 50 of 0.3 µM . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases. Moreover, it is possible that gemcitabine is effective for other untested RNA viruses. Because gemcitabine is a deoxycytidine analog that interferes with DNA as well as RNA synthesis, DNA viruses may not be the exception. Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments.", "Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments. This is especially true for newly emerging or re-emerged viruses involving serious illnesses, such as MERS-CoV, SARS-CoV, and ZIKV, which are major threats to public health and which urgently need an effective treatment during their early stages of infection. In this regard, repurposing of gemcitabine for the treatment of patients infected with these deadly viruses is a realistic approach. Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents.", "Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents. In this manner, an effective antiviral treatment may be achieved by the synergistic action of two antivirals with much lower doses for each drug, which minimizes deleterious side effects when used clinically. As an example, the synergistic antiviral effect of gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few RNA viruses, was reported against EVs such as CVB3 and EV71 . As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials.", "As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials. Even though most antiviral data have originated from in vitro studies, two recent studies have reported the antiviral effects of gemcitabine in murine models . More extensive analyses of gemcitabine in animal models in the near future will accelerate its therapeutic applications in clinical trials. Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis .", "Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis . Moreover, gemcitabine strongly induced the expression of several ISGs including CXCL10, IRF7, IRF9, IFIT1, and DDX58, which were the major effectors in the innate immunity that defended the host against the virus infection. These results were consistent with a previous report that gemcitabine stimulated the production of IFN-β and IFN-γ in IAV-infected RPE cells . Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 6, 10, .", "Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 6, 10, . Regarding purine biosynthesis inhibitors, ribavirin and mycophenolic acid MPA are inhibitors of inosine-5 -monophosphate IMP dehydrogenase IMPDH , which is a key enzyme of the purine biosynthesis pathway. These inhibitors have been successfully used as clinical antiviral or immunosuppressant agents for decades. Both have antiviral activities against viruses such as HCV, hepatitis E virus HEV , MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus WNV , Chikungunya virus CHIKV , and IAV , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses .", "Both have antiviral activities against viruses such as HCV, hepatitis E virus HEV , MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus WNV , Chikungunya virus CHIKV , and IAV , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses . ISG activation occurred through an undefined mechanism that was different from the classical IFN signaling, intracellular dsRNA sensing pathway, Toll-like receptor and nuclear factor B pathways. More importantly, ribavirin-induced ISG activation and antiviral activity were suppressed using supplemented guanosine, a natural analog of ribavirin, suggesting IMPDH inhibition-mediated ISG activation as an alternative innate immunity pathway. Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin .", "Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin . Similar results were obtained with several IMPDH1 or IMPDH2 inhibitors, with various affinities, that were custom-designed and synthesized . As shown in Table 2 , most pyrimidine biosynthesis inhibitors target dihydroorotate dehydrogenase DHODH , an essential enzyme in de novo pyrimidine synthesis. Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element ISRE -stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV .", "Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element ISRE -stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV . DD264 enhanced the expression of several ISGs, which were almost completely suppressed by the addition of supplemented uridine, indicating DHODH inhibition-mediated ISG activation. Moreover, the antiviral activity of and ISG activation by DD264 required the interferon regulatory factor 1 IRF1 transcription factor, a master regulator of antiviral gene expression , which was consistent with the observation that the anti-HCV activity of MPA was partially mediated by IRF1 . In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 .", "In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 . However, whether ISG activation is mediated by pyrimidine biosynthesis inhibition remains to be determined. The mechanism of nucleotide synthesis inhibitor-induced ISG activation is still presently unclear. Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal . First, Wang et al.", "Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal . First, Wang et al. clearly showed that ISG activation and anti-HEV activity induced by MPA or brequinar was not mediated by JAK . Second, IRF7 induction by ribavirin was not affected by knockdown of STAT1, while that of IFN-α was strongly affected under the same conditions . Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine .", "Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine . Moreover, the upregulation of DDX58 mRNAs induced by gemcitabine was not affected by IRF9 knockdown, which was contrary to the result that IFN-α-induced upregulation of DDX58 mRNAs was significantly suppressed under the same conditions. Consistent with above observations, there have been some reports that ISGs was induced in the absence of JAK1 or STAT1 activation . Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the d NTP pool.", "Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the d NTP pool. Inactivation of metabolic enzyme s itself or consequently altered nucleos t ide pools might trigger a signal, which is ultimately delivered to certain cis-acting elements on the promoter of a subset of ISGs, possibly through the relay of kinases and transcription factors. Based on the previously mentioned reports, this signal is less likely to be dependent on STAT1/2-IRF9 IFN-stimulated gene factor 3; ISGF3 , at least for gemcitabine, which is the major transcriptional complex in the IFN-induced JAK/STAT pathway. It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin .", "It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin . Despite the consensus of ISG activation, each purine/pyrimidine biosynthesis inhibitor seems to induce distinct sets of ISGs, at least with different patterns . Targeting an enzyme in which pathways purine or pyrimidine synthesis or steps early/late and de novo/salvage produce different levels of intermediates and nucleos t ides will consequently result in diverse outcomes of ISG activations. There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation.", "There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation. Systematic analyses of signaling kinases, IRFs, and STATs using siRNA knockdown and/or pharmacological inhibition and metabolic analyses of corresponding intermediates and nucleos t ides should therefore clarify the underlying molecular mechanisms of ISG activation by purine/pyrimidine biosynthesis inhibitors. As newly emerging or re-emerged viruses such as SARS-CoV, MERS-CoV, and ZIKV have become a major threat to public health, the need for broad-spectrum antiviral drug has increased. In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs.", "In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs. Nucleoside analogs probably induce different subsets of ISGs, at least with a different pattern, leading to various combinations of ISGs and resulting antiviral outcomes. Moreover, according to Schoggins et al., different viruses are affected by distinct subsets of ISGs and some ISGs such as IRF1, MB21D1, HPSE, DDX58, MDA, and IFITM3 act broadly on various viruses . Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives.", "Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives. Nevertheless, nucleoside analogs interfering with the host nucleotide synthesis pathway suggest possible side effects in their clinical applications. Careful evaluation of clinical safety is required and their application for the urgent measure of patients infected with deadly viruses would be worth being primarily considered." ]	1,594	5,234	Nucleoside analogs have been frequently identified as antiviral agents. In recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. Nucleoside analogs generally interfere with cellular nucleos t ide synthesis pathways, resulting in the depletion or imbalance of d NTP pools. Intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos t ide synthesis inhibition. The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos t ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs. Text: Nucleoside analogs have been historically used for anti-cancer chemotherapy because they inhibit cellular DNA/RNA polymerases . More recently, nucleoside analogs have expanded their therapeutic applications and are being used to develop antiviral drugs against a wide range of serious and life-threatening viruses. Some nucleoside analog drugs targeting specific viral polymerases acyclovir for herpesviruses, zidovudine for human immunodeficiency virus HIV , and sofosbuvir for hepatitis C virus HCV have been successful in clinical trials and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α . Importantly, extensive studies on the antiviral action of ribavirin have established the underlying molecular framework of nucleoside analogs. The primary mechanism to explain the antiviral effect of nucleoside analogs is based on their direct action on viral polymerization. Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferon-stimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV EC 50 of 1.2 µM and 4.9 µM, respectively . More recently, gemcitabine was shown to effectively suppress ZIKV infection and replication in human retinal pigment epithelium RPE cells, particularly at non-cytotoxic concentrations EC 50 of 0.01 µM vs. CC 50 of > 10 µM . ZIKV, a member of the Flaviviridae family, can infect pregnant women and cause congenital abnormalities such as microcephaly in infants, which has attracted increasing public attention as well as extensive research and development into possible treatments. Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively , which were lower concentrations than those used in cancer therapy . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency . In a follow up study, Clouser et al. further reported the antiviral effect of gemcitabine against HIV-related retrovirus, murine leukemia virus MuLV , in vitro EC 50 of 1.6 nM and even in murine AIDS model . A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. EC 50 of 0.068 µM . They also tested whether gemcitabine had an antiviral effect on several other viruses of different families and found its strong inhibitory effect on Sindbis virus and herpes simplex virus-1 HSV-1 >2 log reduction in virus titer but relatively weak effects on Semliki forest virus and human echovirus 6, and minimal effects on Bunyamwera virus, measles virus MeV , and vaccinia virus . The antiviral effect of gemcitabine on EVs, initially performed on Coxsackievirus B3 CVB3 , was found from screening FDA-approved drugs in CVB3 replicon-harboring Vero cells by our group EC 50 of 0.4 µM . Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 EV71 and human rhinoviruses HRVs EC 50 s of 1 and 1-5 µM, respectively . In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model . In this study, intranasal administration of gemcitabine significantly lowered the pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including TNF-α and IL-1β, and the number of lung infiltrating lymphocytes. More recently, Zhang et al. also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells EC 50 of 0.3 µM . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases. Moreover, it is possible that gemcitabine is effective for other untested RNA viruses. Because gemcitabine is a deoxycytidine analog that interferes with DNA as well as RNA synthesis, DNA viruses may not be the exception. Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments. This is especially true for newly emerging or re-emerged viruses involving serious illnesses, such as MERS-CoV, SARS-CoV, and ZIKV, which are major threats to public health and which urgently need an effective treatment during their early stages of infection. In this regard, repurposing of gemcitabine for the treatment of patients infected with these deadly viruses is a realistic approach. Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents. In this manner, an effective antiviral treatment may be achieved by the synergistic action of two antivirals with much lower doses for each drug, which minimizes deleterious side effects when used clinically. As an example, the synergistic antiviral effect of gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few RNA viruses, was reported against EVs such as CVB3 and EV71 . As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials. Even though most antiviral data have originated from in vitro studies, two recent studies have reported the antiviral effects of gemcitabine in murine models . More extensive analyses of gemcitabine in animal models in the near future will accelerate its therapeutic applications in clinical trials. Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis . Moreover, gemcitabine strongly induced the expression of several ISGs including CXCL10, IRF7, IRF9, IFIT1, and DDX58, which were the major effectors in the innate immunity that defended the host against the virus infection. These results were consistent with a previous report that gemcitabine stimulated the production of IFN-β and IFN-γ in IAV-infected RPE cells . Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 6, 10, . Regarding purine biosynthesis inhibitors, ribavirin and mycophenolic acid MPA are inhibitors of inosine-5 -monophosphate IMP dehydrogenase IMPDH , which is a key enzyme of the purine biosynthesis pathway. These inhibitors have been successfully used as clinical antiviral or immunosuppressant agents for decades. Both have antiviral activities against viruses such as HCV, hepatitis E virus HEV , MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus WNV , Chikungunya virus CHIKV , and IAV , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses . ISG activation occurred through an undefined mechanism that was different from the classical IFN signaling, intracellular dsRNA sensing pathway, Toll-like receptor and nuclear factor B pathways. More importantly, ribavirin-induced ISG activation and antiviral activity were suppressed using supplemented guanosine, a natural analog of ribavirin, suggesting IMPDH inhibition-mediated ISG activation as an alternative innate immunity pathway. Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin . Similar results were obtained with several IMPDH1 or IMPDH2 inhibitors, with various affinities, that were custom-designed and synthesized . As shown in Table 2 , most pyrimidine biosynthesis inhibitors target dihydroorotate dehydrogenase DHODH , an essential enzyme in de novo pyrimidine synthesis. Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element ISRE -stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV . DD264 enhanced the expression of several ISGs, which were almost completely suppressed by the addition of supplemented uridine, indicating DHODH inhibition-mediated ISG activation. Moreover, the antiviral activity of and ISG activation by DD264 required the interferon regulatory factor 1 IRF1 transcription factor, a master regulator of antiviral gene expression , which was consistent with the observation that the anti-HCV activity of MPA was partially mediated by IRF1 . In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 . However, whether ISG activation is mediated by pyrimidine biosynthesis inhibition remains to be determined. The mechanism of nucleotide synthesis inhibitor-induced ISG activation is still presently unclear. Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal . First, Wang et al. clearly showed that ISG activation and anti-HEV activity induced by MPA or brequinar was not mediated by JAK . Second, IRF7 induction by ribavirin was not affected by knockdown of STAT1, while that of IFN-α was strongly affected under the same conditions . Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine . Moreover, the upregulation of DDX58 mRNAs induced by gemcitabine was not affected by IRF9 knockdown, which was contrary to the result that IFN-α-induced upregulation of DDX58 mRNAs was significantly suppressed under the same conditions. Consistent with above observations, there have been some reports that ISGs was induced in the absence of JAK1 or STAT1 activation . Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the d NTP pool. Inactivation of metabolic enzyme s itself or consequently altered nucleos t ide pools might trigger a signal, which is ultimately delivered to certain cis-acting elements on the promoter of a subset of ISGs, possibly through the relay of kinases and transcription factors. Based on the previously mentioned reports, this signal is less likely to be dependent on STAT1/2-IRF9 IFN-stimulated gene factor 3; ISGF3 , at least for gemcitabine, which is the major transcriptional complex in the IFN-induced JAK/STAT pathway. It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin . Despite the consensus of ISG activation, each purine/pyrimidine biosynthesis inhibitor seems to induce distinct sets of ISGs, at least with different patterns . Targeting an enzyme in which pathways purine or pyrimidine synthesis or steps early/late and de novo/salvage produce different levels of intermediates and nucleos t ides will consequently result in diverse outcomes of ISG activations. There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation. Systematic analyses of signaling kinases, IRFs, and STATs using siRNA knockdown and/or pharmacological inhibition and metabolic analyses of corresponding intermediates and nucleos t ides should therefore clarify the underlying molecular mechanisms of ISG activation by purine/pyrimidine biosynthesis inhibitors. As newly emerging or re-emerged viruses such as SARS-CoV, MERS-CoV, and ZIKV have become a major threat to public health, the need for broad-spectrum antiviral drug has increased. In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs. Nucleoside analogs probably induce different subsets of ISGs, at least with a different pattern, leading to various combinations of ISGs and resulting antiviral outcomes. Moreover, according to Schoggins et al., different viruses are affected by distinct subsets of ISGs and some ISGs such as IRF1, MB21D1, HPSE, DDX58, MDA, and IFITM3 act broadly on various viruses . Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives. Nevertheless, nucleoside analogs interfering with the host nucleotide synthesis pathway suggest possible side effects in their clinical applications. Careful evaluation of clinical safety is required and their application for the urgent measure of patients infected with deadly viruses would be worth being primarily considered.
How does gemcitabine disrupt viral activity?	by targeting the salvage pathway of pyrimidine biosynthesis	[ "Nucleoside analogs have been frequently identified as antiviral agents. In recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. Nucleoside analogs generally interfere with cellular nucleos t ide synthesis pathways, resulting in the depletion or imbalance of d NTP pools. Intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos t ide synthesis inhibition. The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos t ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs.", "The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos t ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs. Text: Nucleoside analogs have been historically used for anti-cancer chemotherapy because they inhibit cellular DNA/RNA polymerases . More recently, nucleoside analogs have expanded their therapeutic applications and are being used to develop antiviral drugs against a wide range of serious and life-threatening viruses. Some nucleoside analog drugs targeting specific viral polymerases acyclovir for herpesviruses, zidovudine for human immunodeficiency virus HIV , and sofosbuvir for hepatitis C virus HCV have been successful in clinical trials and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α .", "Some nucleoside analog drugs targeting specific viral polymerases acyclovir for herpesviruses, zidovudine for human immunodeficiency virus HIV , and sofosbuvir for hepatitis C virus HCV have been successful in clinical trials and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α . Importantly, extensive studies on the antiviral action of ribavirin have established the underlying molecular framework of nucleoside analogs. The primary mechanism to explain the antiviral effect of nucleoside analogs is based on their direct action on viral polymerization. Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 .", "Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools.", "There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferon-stimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated.", "Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors.", "In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways .", "The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity.", "A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group.", "They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers .", "Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 .", "MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV .", "Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV EC 50 of 1.2 µM and 4.9 µM, respectively .", "To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV EC 50 of 1.2 µM and 4.9 µM, respectively . More recently, gemcitabine was shown to effectively suppress ZIKV infection and replication in human retinal pigment epithelium RPE cells, particularly at non-cytotoxic concentrations EC 50 of 0.01 µM vs. CC 50 of > 10 µM . ZIKV, a member of the Flaviviridae family, can infect pregnant women and cause congenital abnormalities such as microcephaly in infants, which has attracted increasing public attention as well as extensive research and development into possible treatments. Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively , which were lower concentrations than those used in cancer therapy . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency .", "Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively , which were lower concentrations than those used in cancer therapy . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency . In a follow up study, Clouser et al. further reported the antiviral effect of gemcitabine against HIV-related retrovirus, murine leukemia virus MuLV , in vitro EC 50 of 1.6 nM and even in murine AIDS model . A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. EC 50 of 0.068 µM .", "A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. EC 50 of 0.068 µM . They also tested whether gemcitabine had an antiviral effect on several other viruses of different families and found its strong inhibitory effect on Sindbis virus and herpes simplex virus-1 HSV-1 >2 log reduction in virus titer but relatively weak effects on Semliki forest virus and human echovirus 6, and minimal effects on Bunyamwera virus, measles virus MeV , and vaccinia virus . The antiviral effect of gemcitabine on EVs, initially performed on Coxsackievirus B3 CVB3 , was found from screening FDA-approved drugs in CVB3 replicon-harboring Vero cells by our group EC 50 of 0.4 µM . Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 EV71 and human rhinoviruses HRVs EC 50 s of 1 and 1-5 µM, respectively . In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model .", "Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 EV71 and human rhinoviruses HRVs EC 50 s of 1 and 1-5 µM, respectively . In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model . In this study, intranasal administration of gemcitabine significantly lowered the pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including TNF-α and IL-1β, and the number of lung infiltrating lymphocytes. More recently, Zhang et al. also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells EC 50 of 0.3 µM . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases.", "also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells EC 50 of 0.3 µM . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases. Moreover, it is possible that gemcitabine is effective for other untested RNA viruses. Because gemcitabine is a deoxycytidine analog that interferes with DNA as well as RNA synthesis, DNA viruses may not be the exception. Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments.", "Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments. This is especially true for newly emerging or re-emerged viruses involving serious illnesses, such as MERS-CoV, SARS-CoV, and ZIKV, which are major threats to public health and which urgently need an effective treatment during their early stages of infection. In this regard, repurposing of gemcitabine for the treatment of patients infected with these deadly viruses is a realistic approach. Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents.", "Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents. In this manner, an effective antiviral treatment may be achieved by the synergistic action of two antivirals with much lower doses for each drug, which minimizes deleterious side effects when used clinically. As an example, the synergistic antiviral effect of gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few RNA viruses, was reported against EVs such as CVB3 and EV71 . As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials.", "As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials. Even though most antiviral data have originated from in vitro studies, two recent studies have reported the antiviral effects of gemcitabine in murine models . More extensive analyses of gemcitabine in animal models in the near future will accelerate its therapeutic applications in clinical trials. Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis .", "Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis . Moreover, gemcitabine strongly induced the expression of several ISGs including CXCL10, IRF7, IRF9, IFIT1, and DDX58, which were the major effectors in the innate immunity that defended the host against the virus infection. These results were consistent with a previous report that gemcitabine stimulated the production of IFN-β and IFN-γ in IAV-infected RPE cells . Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 6, 10, .", "Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 6, 10, . Regarding purine biosynthesis inhibitors, ribavirin and mycophenolic acid MPA are inhibitors of inosine-5 -monophosphate IMP dehydrogenase IMPDH , which is a key enzyme of the purine biosynthesis pathway. These inhibitors have been successfully used as clinical antiviral or immunosuppressant agents for decades. Both have antiviral activities against viruses such as HCV, hepatitis E virus HEV , MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus WNV , Chikungunya virus CHIKV , and IAV , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses .", "Both have antiviral activities against viruses such as HCV, hepatitis E virus HEV , MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus WNV , Chikungunya virus CHIKV , and IAV , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses . ISG activation occurred through an undefined mechanism that was different from the classical IFN signaling, intracellular dsRNA sensing pathway, Toll-like receptor and nuclear factor B pathways. More importantly, ribavirin-induced ISG activation and antiviral activity were suppressed using supplemented guanosine, a natural analog of ribavirin, suggesting IMPDH inhibition-mediated ISG activation as an alternative innate immunity pathway. Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin .", "Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin . Similar results were obtained with several IMPDH1 or IMPDH2 inhibitors, with various affinities, that were custom-designed and synthesized . As shown in Table 2 , most pyrimidine biosynthesis inhibitors target dihydroorotate dehydrogenase DHODH , an essential enzyme in de novo pyrimidine synthesis. Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element ISRE -stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV .", "Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element ISRE -stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV . DD264 enhanced the expression of several ISGs, which were almost completely suppressed by the addition of supplemented uridine, indicating DHODH inhibition-mediated ISG activation. Moreover, the antiviral activity of and ISG activation by DD264 required the interferon regulatory factor 1 IRF1 transcription factor, a master regulator of antiviral gene expression , which was consistent with the observation that the anti-HCV activity of MPA was partially mediated by IRF1 . In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 .", "In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 . However, whether ISG activation is mediated by pyrimidine biosynthesis inhibition remains to be determined. The mechanism of nucleotide synthesis inhibitor-induced ISG activation is still presently unclear. Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal . First, Wang et al.", "Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal . First, Wang et al. clearly showed that ISG activation and anti-HEV activity induced by MPA or brequinar was not mediated by JAK . Second, IRF7 induction by ribavirin was not affected by knockdown of STAT1, while that of IFN-α was strongly affected under the same conditions . Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine .", "Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine . Moreover, the upregulation of DDX58 mRNAs induced by gemcitabine was not affected by IRF9 knockdown, which was contrary to the result that IFN-α-induced upregulation of DDX58 mRNAs was significantly suppressed under the same conditions. Consistent with above observations, there have been some reports that ISGs was induced in the absence of JAK1 or STAT1 activation . Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the d NTP pool.", "Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the d NTP pool. Inactivation of metabolic enzyme s itself or consequently altered nucleos t ide pools might trigger a signal, which is ultimately delivered to certain cis-acting elements on the promoter of a subset of ISGs, possibly through the relay of kinases and transcription factors. Based on the previously mentioned reports, this signal is less likely to be dependent on STAT1/2-IRF9 IFN-stimulated gene factor 3; ISGF3 , at least for gemcitabine, which is the major transcriptional complex in the IFN-induced JAK/STAT pathway. It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin .", "It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin . Despite the consensus of ISG activation, each purine/pyrimidine biosynthesis inhibitor seems to induce distinct sets of ISGs, at least with different patterns . Targeting an enzyme in which pathways purine or pyrimidine synthesis or steps early/late and de novo/salvage produce different levels of intermediates and nucleos t ides will consequently result in diverse outcomes of ISG activations. There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation.", "There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation. Systematic analyses of signaling kinases, IRFs, and STATs using siRNA knockdown and/or pharmacological inhibition and metabolic analyses of corresponding intermediates and nucleos t ides should therefore clarify the underlying molecular mechanisms of ISG activation by purine/pyrimidine biosynthesis inhibitors. As newly emerging or re-emerged viruses such as SARS-CoV, MERS-CoV, and ZIKV have become a major threat to public health, the need for broad-spectrum antiviral drug has increased. In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs.", "In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs. Nucleoside analogs probably induce different subsets of ISGs, at least with a different pattern, leading to various combinations of ISGs and resulting antiviral outcomes. Moreover, according to Schoggins et al., different viruses are affected by distinct subsets of ISGs and some ISGs such as IRF1, MB21D1, HPSE, DDX58, MDA, and IFITM3 act broadly on various viruses . Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives.", "Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives. Nevertheless, nucleoside analogs interfering with the host nucleotide synthesis pathway suggest possible side effects in their clinical applications. Careful evaluation of clinical safety is required and their application for the urgent measure of patients infected with deadly viruses would be worth being primarily considered." ]	1,594	5,235	Nucleoside analogs have been frequently identified as antiviral agents. In recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. Nucleoside analogs generally interfere with cellular nucleos t ide synthesis pathways, resulting in the depletion or imbalance of d NTP pools. Intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos t ide synthesis inhibition. The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos t ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs. Text: Nucleoside analogs have been historically used for anti-cancer chemotherapy because they inhibit cellular DNA/RNA polymerases . More recently, nucleoside analogs have expanded their therapeutic applications and are being used to develop antiviral drugs against a wide range of serious and life-threatening viruses. Some nucleoside analog drugs targeting specific viral polymerases acyclovir for herpesviruses, zidovudine for human immunodeficiency virus HIV , and sofosbuvir for hepatitis C virus HCV have been successful in clinical trials and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α . Importantly, extensive studies on the antiviral action of ribavirin have established the underlying molecular framework of nucleoside analogs. The primary mechanism to explain the antiviral effect of nucleoside analogs is based on their direct action on viral polymerization. Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferon-stimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV EC 50 of 1.2 µM and 4.9 µM, respectively . More recently, gemcitabine was shown to effectively suppress ZIKV infection and replication in human retinal pigment epithelium RPE cells, particularly at non-cytotoxic concentrations EC 50 of 0.01 µM vs. CC 50 of > 10 µM . ZIKV, a member of the Flaviviridae family, can infect pregnant women and cause congenital abnormalities such as microcephaly in infants, which has attracted increasing public attention as well as extensive research and development into possible treatments. Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively , which were lower concentrations than those used in cancer therapy . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency . In a follow up study, Clouser et al. further reported the antiviral effect of gemcitabine against HIV-related retrovirus, murine leukemia virus MuLV , in vitro EC 50 of 1.6 nM and even in murine AIDS model . A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. EC 50 of 0.068 µM . They also tested whether gemcitabine had an antiviral effect on several other viruses of different families and found its strong inhibitory effect on Sindbis virus and herpes simplex virus-1 HSV-1 >2 log reduction in virus titer but relatively weak effects on Semliki forest virus and human echovirus 6, and minimal effects on Bunyamwera virus, measles virus MeV , and vaccinia virus . The antiviral effect of gemcitabine on EVs, initially performed on Coxsackievirus B3 CVB3 , was found from screening FDA-approved drugs in CVB3 replicon-harboring Vero cells by our group EC 50 of 0.4 µM . Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 EV71 and human rhinoviruses HRVs EC 50 s of 1 and 1-5 µM, respectively . In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model . In this study, intranasal administration of gemcitabine significantly lowered the pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including TNF-α and IL-1β, and the number of lung infiltrating lymphocytes. More recently, Zhang et al. also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells EC 50 of 0.3 µM . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases. Moreover, it is possible that gemcitabine is effective for other untested RNA viruses. Because gemcitabine is a deoxycytidine analog that interferes with DNA as well as RNA synthesis, DNA viruses may not be the exception. Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments. This is especially true for newly emerging or re-emerged viruses involving serious illnesses, such as MERS-CoV, SARS-CoV, and ZIKV, which are major threats to public health and which urgently need an effective treatment during their early stages of infection. In this regard, repurposing of gemcitabine for the treatment of patients infected with these deadly viruses is a realistic approach. Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents. In this manner, an effective antiviral treatment may be achieved by the synergistic action of two antivirals with much lower doses for each drug, which minimizes deleterious side effects when used clinically. As an example, the synergistic antiviral effect of gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few RNA viruses, was reported against EVs such as CVB3 and EV71 . As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials. Even though most antiviral data have originated from in vitro studies, two recent studies have reported the antiviral effects of gemcitabine in murine models . More extensive analyses of gemcitabine in animal models in the near future will accelerate its therapeutic applications in clinical trials. Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis . Moreover, gemcitabine strongly induced the expression of several ISGs including CXCL10, IRF7, IRF9, IFIT1, and DDX58, which were the major effectors in the innate immunity that defended the host against the virus infection. These results were consistent with a previous report that gemcitabine stimulated the production of IFN-β and IFN-γ in IAV-infected RPE cells . Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 6, 10, . Regarding purine biosynthesis inhibitors, ribavirin and mycophenolic acid MPA are inhibitors of inosine-5 -monophosphate IMP dehydrogenase IMPDH , which is a key enzyme of the purine biosynthesis pathway. These inhibitors have been successfully used as clinical antiviral or immunosuppressant agents for decades. Both have antiviral activities against viruses such as HCV, hepatitis E virus HEV , MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus WNV , Chikungunya virus CHIKV , and IAV , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses . ISG activation occurred through an undefined mechanism that was different from the classical IFN signaling, intracellular dsRNA sensing pathway, Toll-like receptor and nuclear factor B pathways. More importantly, ribavirin-induced ISG activation and antiviral activity were suppressed using supplemented guanosine, a natural analog of ribavirin, suggesting IMPDH inhibition-mediated ISG activation as an alternative innate immunity pathway. Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin . Similar results were obtained with several IMPDH1 or IMPDH2 inhibitors, with various affinities, that were custom-designed and synthesized . As shown in Table 2 , most pyrimidine biosynthesis inhibitors target dihydroorotate dehydrogenase DHODH , an essential enzyme in de novo pyrimidine synthesis. Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element ISRE -stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV . DD264 enhanced the expression of several ISGs, which were almost completely suppressed by the addition of supplemented uridine, indicating DHODH inhibition-mediated ISG activation. Moreover, the antiviral activity of and ISG activation by DD264 required the interferon regulatory factor 1 IRF1 transcription factor, a master regulator of antiviral gene expression , which was consistent with the observation that the anti-HCV activity of MPA was partially mediated by IRF1 . In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 . However, whether ISG activation is mediated by pyrimidine biosynthesis inhibition remains to be determined. The mechanism of nucleotide synthesis inhibitor-induced ISG activation is still presently unclear. Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal . First, Wang et al. clearly showed that ISG activation and anti-HEV activity induced by MPA or brequinar was not mediated by JAK . Second, IRF7 induction by ribavirin was not affected by knockdown of STAT1, while that of IFN-α was strongly affected under the same conditions . Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine . Moreover, the upregulation of DDX58 mRNAs induced by gemcitabine was not affected by IRF9 knockdown, which was contrary to the result that IFN-α-induced upregulation of DDX58 mRNAs was significantly suppressed under the same conditions. Consistent with above observations, there have been some reports that ISGs was induced in the absence of JAK1 or STAT1 activation . Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the d NTP pool. Inactivation of metabolic enzyme s itself or consequently altered nucleos t ide pools might trigger a signal, which is ultimately delivered to certain cis-acting elements on the promoter of a subset of ISGs, possibly through the relay of kinases and transcription factors. Based on the previously mentioned reports, this signal is less likely to be dependent on STAT1/2-IRF9 IFN-stimulated gene factor 3; ISGF3 , at least for gemcitabine, which is the major transcriptional complex in the IFN-induced JAK/STAT pathway. It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin . Despite the consensus of ISG activation, each purine/pyrimidine biosynthesis inhibitor seems to induce distinct sets of ISGs, at least with different patterns . Targeting an enzyme in which pathways purine or pyrimidine synthesis or steps early/late and de novo/salvage produce different levels of intermediates and nucleos t ides will consequently result in diverse outcomes of ISG activations. There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation. Systematic analyses of signaling kinases, IRFs, and STATs using siRNA knockdown and/or pharmacological inhibition and metabolic analyses of corresponding intermediates and nucleos t ides should therefore clarify the underlying molecular mechanisms of ISG activation by purine/pyrimidine biosynthesis inhibitors. As newly emerging or re-emerged viruses such as SARS-CoV, MERS-CoV, and ZIKV have become a major threat to public health, the need for broad-spectrum antiviral drug has increased. In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs. Nucleoside analogs probably induce different subsets of ISGs, at least with a different pattern, leading to various combinations of ISGs and resulting antiviral outcomes. Moreover, according to Schoggins et al., different viruses are affected by distinct subsets of ISGs and some ISGs such as IRF1, MB21D1, HPSE, DDX58, MDA, and IFITM3 act broadly on various viruses . Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives. Nevertheless, nucleoside analogs interfering with the host nucleotide synthesis pathway suggest possible side effects in their clinical applications. Careful evaluation of clinical safety is required and their application for the urgent measure of patients infected with deadly viruses would be worth being primarily considered.
What is the main cause of death in the neonatal period of calves?	Calf septicemia	[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]	1,560	2,129	AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.
Where was hepcidin first discovered?	human urine	[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]	1,560	2,131	AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.
What is hepcidin?	low molecular weight, antimicrobial peptide hormone	[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]	1,560	2,130	AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.
What organ produces hepcidin?	liver	[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]	1,560	2,132	AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.
What stimulates the release of hepcidin?	inflammatory reactions and high Fe concentrations	[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]	1,560	2,133	AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.
What element does hepcidin play a roles in regulating during metabolism?	Fe	[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]	1,560	2,134	AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.
Is hepcidin toxic?	potentially toxic	[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]	1,560	2,135	AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.
Why is iron critical to bacteria?	bacteria utilize Fe for survival, growth and proliferation	[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]	1,560	2,136	AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.
How does hepcidin work in the duodenum?	control of excessive Fe absorption	[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]	1,560	2,137	AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.
How does hepcidin affect macrophages?	regulation of Fe release	[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]	1,560	2,138	AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.
What leads to oxidative stress in the body?	production of ROS	[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]	1,560	2,139	AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.
What parameter is used to measure antioxidant levels?	superoxide dismutase	[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]	1,560	2,140	AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.
What is Acute hemorrhagic encephalomyelitis?	a rare form of acute disseminated encephalomyelitis	[ "BACKGROUND: Acute hemorrhagic encephalomyelitis AHEM is considered as a rare form of acute disseminated encephalomyelitis characterized by fulminant encephalopathy with hemorrhagic necrosis and most often fatal outcome. OBJECTIVE: To report the association with Ran Binding Protein RANBP2 gene variant and the response to decompressive craniectomy and high-dose intravenous methylprednisolone IVMP in life-threatening AHEM. DESIGN: Single case study. CASE REPORT: A 6-year-old girl known to have sickle cell disease SCD presented an acquired demyelinating syndrome ADS with diplopia due to sudden unilateral fourth nerve palsy. She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma.", "She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma. Brain magnetic resonance imaging showed a left frontal necrotico-hemorrhagic lesion and new multifocal areas of demyelination. She underwent decompressive craniotomy and evacuation of an ongoing left frontoparietal hemorrhage. Comprehensive investigations ruled out vascular and infectious process. The neurological deterioration stopped concomitantly with combined neurosurgical drainage of the hematoma, decompressive craniotomy, IVMP, and intravenous immunoglobulins IVIG . She developed during the following months Crohn disease and sclerosing cholangitis. After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered.", "After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered. A heterozygous novel rare missense variant c.4993A>G, p.Lys1665Glu was identified in RANBP2, a gene associated with acute necrotizing encephalopathy. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP.", "RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP. This case report, in addition to others, stresses the likely efficacy of combined craniotomy, IVIG, and IVMP treatments in AHEM. RANBP2 mutations may sensitize the brain to inflammation and predispose to AHEM. Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system.", "Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system. It is usually fatal . . . . Many treatment options have been used including intravenous IV steroids, intravenous immunoglobulins IVIG , and plasmapheresis . . There have been few reports of survival following early intervention with high-dose corticosteroid therapy and/or decompressive craniotomy . . . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle.", ". . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle. RANBP2 is associated with microtubules and mitochondria suggesting roles in intracellular protein trafficking or energy maintenance and homeostasis of neuronal cells. RANBP2 mutations have been reported in acute necrotizing encephalopathy ANE which could present with coma, convulsions, and encephalopathy. The hallmark of ANE is multiple, symmetric brain lesions located in the thalami bilaterally, putamina, deep periventricular white matter, cerebellum, and brainstem. It could be triggered by a viral infection in previously healthy children . .", "It could be triggered by a viral infection in previously healthy children . . We report a new case of AHEM associated to a Ran Binding Protein RANBP -2 variant and responsive to combined craniectomy, intravenous methylprednisolone IVMP , and IVIG as inaugural manifestation of multisystemic autoimmunity in a girl with sickle cell disease SCD . A 6-year-old girl known for SCD treated on folic acid and hydroxyurea was admitted for new-onset diplopia day 0 D0 : refers to the start of the diplopia 6 weeks after respiratory tract infection due to rhinovirus. She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 .", "She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 . Brain MR angiography MRA showed a normal appearing circle of Willis. The cerebrospinal fluid CSF obtained by lumber puncture was normal WBC 1 cells/μl, RBC 0 cells/μl, glucose 2.9 mmol/L, protein 0.18 g/L, and absent oligoclonal bands . The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area.", "The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area. Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h.", "Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h. She was treated with five doses of IVMP 30 mg/kg/day followed by 9 days of oral prednisone 1 mg/kg/day . At discharge, her neurological exam was significant only for vertical diplopia. She presented 1 month later with 5 days of upper respiratory tract infection symptoms, fever, headache, and a rapidly progressive right-hand weakness D30 with normal alertness. She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir.", "She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir. White cell count was 13.4 × 10 9 /L, hemoglobin was 7.8 g/L, and platelets were 239 × 10 9 /L. While in the MRI machine D30 she deteriorated with vomiting and reduced level of consciousness Glasgow Coma Scale dropped from 15 to 8 over 30 min . Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F .", "Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F . The patient was immediately brought out of the magnet and her physical exam demonstrated unequal dilated pupils. She received IV mannitol and hypertonic saline for the management of acute intracranial hypertension/ herniation and was taken for surgery. She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis.", "She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis. Estimated blood loss was 3.5 L. She received 8 units of packed red blood cells, 3 units of cryoprecipitate, 6 units of fresh frozen plasma, and 3 units of platelets. Coagulation profile showed international normalization ratio = 3.38, prothrombin time = 51.2 s, and partial thromboplastin time = 122 s. An intraventricular pressure monitor was inserted. She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces.", "She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces. New white matter lesions were detected in the left posterior parietal and occipital lobes and in the left caudate head. MRI at D33 showed interval worsening with disseminated gray and white matter non-hemorrhagic lesions in the right cerebral and both cerebellar hemispheres, bilateral deep gray nuclei, as well as new necrotic non-hemorrhagic lesions in the left hemisphere Figures 2G-I . She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 .", "She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 . Prednisolone was tapered course over 6 weeks. At discharge D71 , she was able to say a few words and had better power of her right side. Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma.", "Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma. There was diffuse microglial activation and signs of early microinfarcts. Blood, CSF and urine culture, and PCR HSV1/2 were negative for bacteria and for viruses. CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative.", "CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative. Anti-extractable nuclear antigens SSA-RO, SSB-LA, smith, RNP were negative. Serum autoimmune antibodies panel NMO, NMDAR, AMPA I/II, GAB, MAG, VGCC, MOG, YO, HU, RI were negative but GAD antibody was slightly positive, possibly due to the IVIG infusion. EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange.", "EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange. Six months later, the patient was diagnosed to have Crohn's disease and primary sclerosing cholangitis. Two years later, the patient still suffers right hemiparesis but is able to walk without support. She presents an expressive aphasia. Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu .", "Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu . This mutation has not been previously reported in the HGMD database. This variant has been observed at a frequency of <0.01% across the entire Broad ExAC dataset of individuals without severe childhood onset disease 6/117,118 alleles . Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD .", "Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD . Several differential diagnoses of acute encephalopathy in a patient with sickle cell anemia can be considered. An infectious encephalitis, including herpes encephalitis, was ruled out by blood and CSF bacterial and viral cultures and negative HSV I/ II PCR. Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . .", "Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . . SCD patients are prone to ischemic or hemorrhagic strokes . . Primary hemorrhagic stroke is uncommon in pediatric SCD. Most cases were from adults and have been described in the context of previous ischemic stroke, aneurysms, low hemoglobin, acute chest syndrome, and hypertransfusions. Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . .", "Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . . This was ruled out as the MRI findings were not consistent with a specific vascular territory and normal arterial and venous flows were shown on vascular imaging. Another differential is posterior reversible encephalopathy syndrome which has been reported in SCD patients . . . . . However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury.", "However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury. However, it is associated to high ferritin and low triglycerides at the time of the encephalopathy, other multisystemic injuries, typical neuropathological findings, and recurrence over time, which were not noted in our patient . . Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia.", ". Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia. It caused punctate areas of hemorrhages in the basal ganglia, periventricular white matter, and mainly along the posterior parietal cortex. This was attributed to parvovirus B19-induced vasculitis . . In our patient, there was no sign of aplasia or any neuroradiological finding of parvovirus B19 infection. Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . .", "Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . . This was ruled out as the patient did not have clinical or radiological signs of acute chest syndrome or embolism and there was no arterial hypoxemia. Acute hemorrhagic encephalomyelitis has been described in pediatric patients following ADEM or ADEM-like episodes . . AHEM is the most plausible diagnosis in our patients based on the clinical and radiological presentation, the preceding ADEM-like episode, and the exclusion of other etiologies of acute encephalopathy. Other patients with AHEM have been described in the SCD context . .", "Other patients with AHEM have been described in the SCD context . . Many treatment options have been used to treat AHEM; of these, IV steroids have been associated with survival following aggressive, high-dose corticosteroid therapy . . . . . . . . . . Autosomal dominant mutations with incomplete penetrance in RANBP2 have been associated with susceptibility to infectioninduced necrotizing encephalopathy . . Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . .", "Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . . Our patient has a similar presentation and imaging features as infection-induced necrotizing encephalopathy, including bilateral thalamic involvement. The rare heterozygous previously unreported variant we identified in RANBP2 affects a very conserved aminoacid and is predicted deleterious using in silico tools a prediction tool performing a fast bioinformatics analysis which can predict the pathogenicity of a variant based on the change to an amino acid . It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy .", "It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy . making possible that both entities might be part of the same pathophysiological continuum. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells . . Hence, RANBP2 dysfunction might make neuronal cells much vulnerable to energy failure and necrosis when exposed to inflammatory or other stresses, such as those implicated in AHEM. This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication.", "This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication. All authors participated in gathering the data, designing the article, and discussing and editing the manuscript. aCKNoWleDgMeNts We thank Dr. S. Abish, Dr. N. Ahmed, and Mrs. C. Guiraut for their help. We are grateful to the Hoppenheim Fund from the Montreal Children Hospital Foundation. The first author of this article received a scholarship from the Hoppenheim Fund, Montreal Children Hospital Foundation .. This work was supported by grants from Heart and Stroke Foundation of Canada grant number: G-14-0005756 , and Foundation of Stars." ]	1,561	3,032	BACKGROUND: Acute hemorrhagic encephalomyelitis AHEM is considered as a rare form of acute disseminated encephalomyelitis characterized by fulminant encephalopathy with hemorrhagic necrosis and most often fatal outcome. OBJECTIVE: To report the association with Ran Binding Protein RANBP2 gene variant and the response to decompressive craniectomy and high-dose intravenous methylprednisolone IVMP in life-threatening AHEM. DESIGN: Single case study. CASE REPORT: A 6-year-old girl known to have sickle cell disease SCD presented an acquired demyelinating syndrome ADS with diplopia due to sudden unilateral fourth nerve palsy. She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma. Brain magnetic resonance imaging showed a left frontal necrotico-hemorrhagic lesion and new multifocal areas of demyelination. She underwent decompressive craniotomy and evacuation of an ongoing left frontoparietal hemorrhage. Comprehensive investigations ruled out vascular and infectious process. The neurological deterioration stopped concomitantly with combined neurosurgical drainage of the hematoma, decompressive craniotomy, IVMP, and intravenous immunoglobulins IVIG . She developed during the following months Crohn disease and sclerosing cholangitis. After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered. A heterozygous novel rare missense variant c.4993A>G, p.Lys1665Glu was identified in RANBP2, a gene associated with acute necrotizing encephalopathy. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP. This case report, in addition to others, stresses the likely efficacy of combined craniotomy, IVIG, and IVMP treatments in AHEM. RANBP2 mutations may sensitize the brain to inflammation and predispose to AHEM. Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system. It is usually fatal . . . . Many treatment options have been used including intravenous IV steroids, intravenous immunoglobulins IVIG , and plasmapheresis . . There have been few reports of survival following early intervention with high-dose corticosteroid therapy and/or decompressive craniotomy . . . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle. RANBP2 is associated with microtubules and mitochondria suggesting roles in intracellular protein trafficking or energy maintenance and homeostasis of neuronal cells. RANBP2 mutations have been reported in acute necrotizing encephalopathy ANE which could present with coma, convulsions, and encephalopathy. The hallmark of ANE is multiple, symmetric brain lesions located in the thalami bilaterally, putamina, deep periventricular white matter, cerebellum, and brainstem. It could be triggered by a viral infection in previously healthy children . . We report a new case of AHEM associated to a Ran Binding Protein RANBP -2 variant and responsive to combined craniectomy, intravenous methylprednisolone IVMP , and IVIG as inaugural manifestation of multisystemic autoimmunity in a girl with sickle cell disease SCD . A 6-year-old girl known for SCD treated on folic acid and hydroxyurea was admitted for new-onset diplopia day 0 D0 : refers to the start of the diplopia 6 weeks after respiratory tract infection due to rhinovirus. She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 . Brain MR angiography MRA showed a normal appearing circle of Willis. The cerebrospinal fluid CSF obtained by lumber puncture was normal WBC 1 cells/μl, RBC 0 cells/μl, glucose 2.9 mmol/L, protein 0.18 g/L, and absent oligoclonal bands . The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area. Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h. She was treated with five doses of IVMP 30 mg/kg/day followed by 9 days of oral prednisone 1 mg/kg/day . At discharge, her neurological exam was significant only for vertical diplopia. She presented 1 month later with 5 days of upper respiratory tract infection symptoms, fever, headache, and a rapidly progressive right-hand weakness D30 with normal alertness. She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir. White cell count was 13.4 × 10 9 /L, hemoglobin was 7.8 g/L, and platelets were 239 × 10 9 /L. While in the MRI machine D30 she deteriorated with vomiting and reduced level of consciousness Glasgow Coma Scale dropped from 15 to 8 over 30 min . Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F . The patient was immediately brought out of the magnet and her physical exam demonstrated unequal dilated pupils. She received IV mannitol and hypertonic saline for the management of acute intracranial hypertension/ herniation and was taken for surgery. She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis. Estimated blood loss was 3.5 L. She received 8 units of packed red blood cells, 3 units of cryoprecipitate, 6 units of fresh frozen plasma, and 3 units of platelets. Coagulation profile showed international normalization ratio = 3.38, prothrombin time = 51.2 s, and partial thromboplastin time = 122 s. An intraventricular pressure monitor was inserted. She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces. New white matter lesions were detected in the left posterior parietal and occipital lobes and in the left caudate head. MRI at D33 showed interval worsening with disseminated gray and white matter non-hemorrhagic lesions in the right cerebral and both cerebellar hemispheres, bilateral deep gray nuclei, as well as new necrotic non-hemorrhagic lesions in the left hemisphere Figures 2G-I . She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 . Prednisolone was tapered course over 6 weeks. At discharge D71 , she was able to say a few words and had better power of her right side. Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma. There was diffuse microglial activation and signs of early microinfarcts. Blood, CSF and urine culture, and PCR HSV1/2 were negative for bacteria and for viruses. CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative. Anti-extractable nuclear antigens SSA-RO, SSB-LA, smith, RNP were negative. Serum autoimmune antibodies panel NMO, NMDAR, AMPA I/II, GAB, MAG, VGCC, MOG, YO, HU, RI were negative but GAD antibody was slightly positive, possibly due to the IVIG infusion. EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange. Six months later, the patient was diagnosed to have Crohn's disease and primary sclerosing cholangitis. Two years later, the patient still suffers right hemiparesis but is able to walk without support. She presents an expressive aphasia. Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu . This mutation has not been previously reported in the HGMD database. This variant has been observed at a frequency of <0.01% across the entire Broad ExAC dataset of individuals without severe childhood onset disease 6/117,118 alleles . Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD . Several differential diagnoses of acute encephalopathy in a patient with sickle cell anemia can be considered. An infectious encephalitis, including herpes encephalitis, was ruled out by blood and CSF bacterial and viral cultures and negative HSV I/ II PCR. Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . . SCD patients are prone to ischemic or hemorrhagic strokes . . Primary hemorrhagic stroke is uncommon in pediatric SCD. Most cases were from adults and have been described in the context of previous ischemic stroke, aneurysms, low hemoglobin, acute chest syndrome, and hypertransfusions. Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . . This was ruled out as the MRI findings were not consistent with a specific vascular territory and normal arterial and venous flows were shown on vascular imaging. Another differential is posterior reversible encephalopathy syndrome which has been reported in SCD patients . . . . . However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury. However, it is associated to high ferritin and low triglycerides at the time of the encephalopathy, other multisystemic injuries, typical neuropathological findings, and recurrence over time, which were not noted in our patient . . Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia. It caused punctate areas of hemorrhages in the basal ganglia, periventricular white matter, and mainly along the posterior parietal cortex. This was attributed to parvovirus B19-induced vasculitis . . In our patient, there was no sign of aplasia or any neuroradiological finding of parvovirus B19 infection. Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . . This was ruled out as the patient did not have clinical or radiological signs of acute chest syndrome or embolism and there was no arterial hypoxemia. Acute hemorrhagic encephalomyelitis has been described in pediatric patients following ADEM or ADEM-like episodes . . AHEM is the most plausible diagnosis in our patients based on the clinical and radiological presentation, the preceding ADEM-like episode, and the exclusion of other etiologies of acute encephalopathy. Other patients with AHEM have been described in the SCD context . . Many treatment options have been used to treat AHEM; of these, IV steroids have been associated with survival following aggressive, high-dose corticosteroid therapy . . . . . . . . . . Autosomal dominant mutations with incomplete penetrance in RANBP2 have been associated with susceptibility to infectioninduced necrotizing encephalopathy . . Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . . Our patient has a similar presentation and imaging features as infection-induced necrotizing encephalopathy, including bilateral thalamic involvement. The rare heterozygous previously unreported variant we identified in RANBP2 affects a very conserved aminoacid and is predicted deleterious using in silico tools a prediction tool performing a fast bioinformatics analysis which can predict the pathogenicity of a variant based on the change to an amino acid . It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy . making possible that both entities might be part of the same pathophysiological continuum. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells . . Hence, RANBP2 dysfunction might make neuronal cells much vulnerable to energy failure and necrosis when exposed to inflammatory or other stresses, such as those implicated in AHEM. This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication. All authors participated in gathering the data, designing the article, and discussing and editing the manuscript. aCKNoWleDgMeNts We thank Dr. S. Abish, Dr. N. Ahmed, and Mrs. C. Guiraut for their help. We are grateful to the Hoppenheim Fund from the Montreal Children Hospital Foundation. The first author of this article received a scholarship from the Hoppenheim Fund, Montreal Children Hospital Foundation .. This work was supported by grants from Heart and Stroke Foundation of Canada grant number: G-14-0005756 , and Foundation of Stars.
What are the salient findings in Acute hemorrhagic encephalomyelitis?	fulminant encephalopathy with hemorrhagic necrosis	[ "BACKGROUND: Acute hemorrhagic encephalomyelitis AHEM is considered as a rare form of acute disseminated encephalomyelitis characterized by fulminant encephalopathy with hemorrhagic necrosis and most often fatal outcome. OBJECTIVE: To report the association with Ran Binding Protein RANBP2 gene variant and the response to decompressive craniectomy and high-dose intravenous methylprednisolone IVMP in life-threatening AHEM. DESIGN: Single case study. CASE REPORT: A 6-year-old girl known to have sickle cell disease SCD presented an acquired demyelinating syndrome ADS with diplopia due to sudden unilateral fourth nerve palsy. She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma.", "She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma. Brain magnetic resonance imaging showed a left frontal necrotico-hemorrhagic lesion and new multifocal areas of demyelination. She underwent decompressive craniotomy and evacuation of an ongoing left frontoparietal hemorrhage. Comprehensive investigations ruled out vascular and infectious process. The neurological deterioration stopped concomitantly with combined neurosurgical drainage of the hematoma, decompressive craniotomy, IVMP, and intravenous immunoglobulins IVIG . She developed during the following months Crohn disease and sclerosing cholangitis. After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered.", "After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered. A heterozygous novel rare missense variant c.4993A>G, p.Lys1665Glu was identified in RANBP2, a gene associated with acute necrotizing encephalopathy. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP.", "RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP. This case report, in addition to others, stresses the likely efficacy of combined craniotomy, IVIG, and IVMP treatments in AHEM. RANBP2 mutations may sensitize the brain to inflammation and predispose to AHEM. Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system.", "Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system. It is usually fatal . . . . Many treatment options have been used including intravenous IV steroids, intravenous immunoglobulins IVIG , and plasmapheresis . . There have been few reports of survival following early intervention with high-dose corticosteroid therapy and/or decompressive craniotomy . . . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle.", ". . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle. RANBP2 is associated with microtubules and mitochondria suggesting roles in intracellular protein trafficking or energy maintenance and homeostasis of neuronal cells. RANBP2 mutations have been reported in acute necrotizing encephalopathy ANE which could present with coma, convulsions, and encephalopathy. The hallmark of ANE is multiple, symmetric brain lesions located in the thalami bilaterally, putamina, deep periventricular white matter, cerebellum, and brainstem. It could be triggered by a viral infection in previously healthy children . .", "It could be triggered by a viral infection in previously healthy children . . We report a new case of AHEM associated to a Ran Binding Protein RANBP -2 variant and responsive to combined craniectomy, intravenous methylprednisolone IVMP , and IVIG as inaugural manifestation of multisystemic autoimmunity in a girl with sickle cell disease SCD . A 6-year-old girl known for SCD treated on folic acid and hydroxyurea was admitted for new-onset diplopia day 0 D0 : refers to the start of the diplopia 6 weeks after respiratory tract infection due to rhinovirus. She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 .", "She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 . Brain MR angiography MRA showed a normal appearing circle of Willis. The cerebrospinal fluid CSF obtained by lumber puncture was normal WBC 1 cells/μl, RBC 0 cells/μl, glucose 2.9 mmol/L, protein 0.18 g/L, and absent oligoclonal bands . The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area.", "The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area. Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h.", "Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h. She was treated with five doses of IVMP 30 mg/kg/day followed by 9 days of oral prednisone 1 mg/kg/day . At discharge, her neurological exam was significant only for vertical diplopia. She presented 1 month later with 5 days of upper respiratory tract infection symptoms, fever, headache, and a rapidly progressive right-hand weakness D30 with normal alertness. She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir.", "She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir. White cell count was 13.4 × 10 9 /L, hemoglobin was 7.8 g/L, and platelets were 239 × 10 9 /L. While in the MRI machine D30 she deteriorated with vomiting and reduced level of consciousness Glasgow Coma Scale dropped from 15 to 8 over 30 min . Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F .", "Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F . The patient was immediately brought out of the magnet and her physical exam demonstrated unequal dilated pupils. She received IV mannitol and hypertonic saline for the management of acute intracranial hypertension/ herniation and was taken for surgery. She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis.", "She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis. Estimated blood loss was 3.5 L. She received 8 units of packed red blood cells, 3 units of cryoprecipitate, 6 units of fresh frozen plasma, and 3 units of platelets. Coagulation profile showed international normalization ratio = 3.38, prothrombin time = 51.2 s, and partial thromboplastin time = 122 s. An intraventricular pressure monitor was inserted. She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces.", "She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces. New white matter lesions were detected in the left posterior parietal and occipital lobes and in the left caudate head. MRI at D33 showed interval worsening with disseminated gray and white matter non-hemorrhagic lesions in the right cerebral and both cerebellar hemispheres, bilateral deep gray nuclei, as well as new necrotic non-hemorrhagic lesions in the left hemisphere Figures 2G-I . She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 .", "She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 . Prednisolone was tapered course over 6 weeks. At discharge D71 , she was able to say a few words and had better power of her right side. Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma.", "Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma. There was diffuse microglial activation and signs of early microinfarcts. Blood, CSF and urine culture, and PCR HSV1/2 were negative for bacteria and for viruses. CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative.", "CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative. Anti-extractable nuclear antigens SSA-RO, SSB-LA, smith, RNP were negative. Serum autoimmune antibodies panel NMO, NMDAR, AMPA I/II, GAB, MAG, VGCC, MOG, YO, HU, RI were negative but GAD antibody was slightly positive, possibly due to the IVIG infusion. EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange.", "EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange. Six months later, the patient was diagnosed to have Crohn's disease and primary sclerosing cholangitis. Two years later, the patient still suffers right hemiparesis but is able to walk without support. She presents an expressive aphasia. Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu .", "Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu . This mutation has not been previously reported in the HGMD database. This variant has been observed at a frequency of <0.01% across the entire Broad ExAC dataset of individuals without severe childhood onset disease 6/117,118 alleles . Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD .", "Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD . Several differential diagnoses of acute encephalopathy in a patient with sickle cell anemia can be considered. An infectious encephalitis, including herpes encephalitis, was ruled out by blood and CSF bacterial and viral cultures and negative HSV I/ II PCR. Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . .", "Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . . SCD patients are prone to ischemic or hemorrhagic strokes . . Primary hemorrhagic stroke is uncommon in pediatric SCD. Most cases were from adults and have been described in the context of previous ischemic stroke, aneurysms, low hemoglobin, acute chest syndrome, and hypertransfusions. Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . .", "Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . . This was ruled out as the MRI findings were not consistent with a specific vascular territory and normal arterial and venous flows were shown on vascular imaging. Another differential is posterior reversible encephalopathy syndrome which has been reported in SCD patients . . . . . However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury.", "However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury. However, it is associated to high ferritin and low triglycerides at the time of the encephalopathy, other multisystemic injuries, typical neuropathological findings, and recurrence over time, which were not noted in our patient . . Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia.", ". Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia. It caused punctate areas of hemorrhages in the basal ganglia, periventricular white matter, and mainly along the posterior parietal cortex. This was attributed to parvovirus B19-induced vasculitis . . In our patient, there was no sign of aplasia or any neuroradiological finding of parvovirus B19 infection. Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . .", "Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . . This was ruled out as the patient did not have clinical or radiological signs of acute chest syndrome or embolism and there was no arterial hypoxemia. Acute hemorrhagic encephalomyelitis has been described in pediatric patients following ADEM or ADEM-like episodes . . AHEM is the most plausible diagnosis in our patients based on the clinical and radiological presentation, the preceding ADEM-like episode, and the exclusion of other etiologies of acute encephalopathy. Other patients with AHEM have been described in the SCD context . .", "Other patients with AHEM have been described in the SCD context . . Many treatment options have been used to treat AHEM; of these, IV steroids have been associated with survival following aggressive, high-dose corticosteroid therapy . . . . . . . . . . Autosomal dominant mutations with incomplete penetrance in RANBP2 have been associated with susceptibility to infectioninduced necrotizing encephalopathy . . Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . .", "Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . . Our patient has a similar presentation and imaging features as infection-induced necrotizing encephalopathy, including bilateral thalamic involvement. The rare heterozygous previously unreported variant we identified in RANBP2 affects a very conserved aminoacid and is predicted deleterious using in silico tools a prediction tool performing a fast bioinformatics analysis which can predict the pathogenicity of a variant based on the change to an amino acid . It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy .", "It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy . making possible that both entities might be part of the same pathophysiological continuum. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells . . Hence, RANBP2 dysfunction might make neuronal cells much vulnerable to energy failure and necrosis when exposed to inflammatory or other stresses, such as those implicated in AHEM. This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication.", "This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication. All authors participated in gathering the data, designing the article, and discussing and editing the manuscript. aCKNoWleDgMeNts We thank Dr. S. Abish, Dr. N. Ahmed, and Mrs. C. Guiraut for their help. We are grateful to the Hoppenheim Fund from the Montreal Children Hospital Foundation. The first author of this article received a scholarship from the Hoppenheim Fund, Montreal Children Hospital Foundation .. This work was supported by grants from Heart and Stroke Foundation of Canada grant number: G-14-0005756 , and Foundation of Stars." ]	1,561	3,033	BACKGROUND: Acute hemorrhagic encephalomyelitis AHEM is considered as a rare form of acute disseminated encephalomyelitis characterized by fulminant encephalopathy with hemorrhagic necrosis and most often fatal outcome. OBJECTIVE: To report the association with Ran Binding Protein RANBP2 gene variant and the response to decompressive craniectomy and high-dose intravenous methylprednisolone IVMP in life-threatening AHEM. DESIGN: Single case study. CASE REPORT: A 6-year-old girl known to have sickle cell disease SCD presented an acquired demyelinating syndrome ADS with diplopia due to sudden unilateral fourth nerve palsy. She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma. Brain magnetic resonance imaging showed a left frontal necrotico-hemorrhagic lesion and new multifocal areas of demyelination. She underwent decompressive craniotomy and evacuation of an ongoing left frontoparietal hemorrhage. Comprehensive investigations ruled out vascular and infectious process. The neurological deterioration stopped concomitantly with combined neurosurgical drainage of the hematoma, decompressive craniotomy, IVMP, and intravenous immunoglobulins IVIG . She developed during the following months Crohn disease and sclerosing cholangitis. After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered. A heterozygous novel rare missense variant c.4993A>G, p.Lys1665Glu was identified in RANBP2, a gene associated with acute necrotizing encephalopathy. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP. This case report, in addition to others, stresses the likely efficacy of combined craniotomy, IVIG, and IVMP treatments in AHEM. RANBP2 mutations may sensitize the brain to inflammation and predispose to AHEM. Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system. It is usually fatal . . . . Many treatment options have been used including intravenous IV steroids, intravenous immunoglobulins IVIG , and plasmapheresis . . There have been few reports of survival following early intervention with high-dose corticosteroid therapy and/or decompressive craniotomy . . . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle. RANBP2 is associated with microtubules and mitochondria suggesting roles in intracellular protein trafficking or energy maintenance and homeostasis of neuronal cells. RANBP2 mutations have been reported in acute necrotizing encephalopathy ANE which could present with coma, convulsions, and encephalopathy. The hallmark of ANE is multiple, symmetric brain lesions located in the thalami bilaterally, putamina, deep periventricular white matter, cerebellum, and brainstem. It could be triggered by a viral infection in previously healthy children . . We report a new case of AHEM associated to a Ran Binding Protein RANBP -2 variant and responsive to combined craniectomy, intravenous methylprednisolone IVMP , and IVIG as inaugural manifestation of multisystemic autoimmunity in a girl with sickle cell disease SCD . A 6-year-old girl known for SCD treated on folic acid and hydroxyurea was admitted for new-onset diplopia day 0 D0 : refers to the start of the diplopia 6 weeks after respiratory tract infection due to rhinovirus. She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 . Brain MR angiography MRA showed a normal appearing circle of Willis. The cerebrospinal fluid CSF obtained by lumber puncture was normal WBC 1 cells/μl, RBC 0 cells/μl, glucose 2.9 mmol/L, protein 0.18 g/L, and absent oligoclonal bands . The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area. Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h. She was treated with five doses of IVMP 30 mg/kg/day followed by 9 days of oral prednisone 1 mg/kg/day . At discharge, her neurological exam was significant only for vertical diplopia. She presented 1 month later with 5 days of upper respiratory tract infection symptoms, fever, headache, and a rapidly progressive right-hand weakness D30 with normal alertness. She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir. White cell count was 13.4 × 10 9 /L, hemoglobin was 7.8 g/L, and platelets were 239 × 10 9 /L. While in the MRI machine D30 she deteriorated with vomiting and reduced level of consciousness Glasgow Coma Scale dropped from 15 to 8 over 30 min . Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F . The patient was immediately brought out of the magnet and her physical exam demonstrated unequal dilated pupils. She received IV mannitol and hypertonic saline for the management of acute intracranial hypertension/ herniation and was taken for surgery. She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis. Estimated blood loss was 3.5 L. She received 8 units of packed red blood cells, 3 units of cryoprecipitate, 6 units of fresh frozen plasma, and 3 units of platelets. Coagulation profile showed international normalization ratio = 3.38, prothrombin time = 51.2 s, and partial thromboplastin time = 122 s. An intraventricular pressure monitor was inserted. She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces. New white matter lesions were detected in the left posterior parietal and occipital lobes and in the left caudate head. MRI at D33 showed interval worsening with disseminated gray and white matter non-hemorrhagic lesions in the right cerebral and both cerebellar hemispheres, bilateral deep gray nuclei, as well as new necrotic non-hemorrhagic lesions in the left hemisphere Figures 2G-I . She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 . Prednisolone was tapered course over 6 weeks. At discharge D71 , she was able to say a few words and had better power of her right side. Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma. There was diffuse microglial activation and signs of early microinfarcts. Blood, CSF and urine culture, and PCR HSV1/2 were negative for bacteria and for viruses. CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative. Anti-extractable nuclear antigens SSA-RO, SSB-LA, smith, RNP were negative. Serum autoimmune antibodies panel NMO, NMDAR, AMPA I/II, GAB, MAG, VGCC, MOG, YO, HU, RI were negative but GAD antibody was slightly positive, possibly due to the IVIG infusion. EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange. Six months later, the patient was diagnosed to have Crohn's disease and primary sclerosing cholangitis. Two years later, the patient still suffers right hemiparesis but is able to walk without support. She presents an expressive aphasia. Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu . This mutation has not been previously reported in the HGMD database. This variant has been observed at a frequency of <0.01% across the entire Broad ExAC dataset of individuals without severe childhood onset disease 6/117,118 alleles . Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD . Several differential diagnoses of acute encephalopathy in a patient with sickle cell anemia can be considered. An infectious encephalitis, including herpes encephalitis, was ruled out by blood and CSF bacterial and viral cultures and negative HSV I/ II PCR. Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . . SCD patients are prone to ischemic or hemorrhagic strokes . . Primary hemorrhagic stroke is uncommon in pediatric SCD. Most cases were from adults and have been described in the context of previous ischemic stroke, aneurysms, low hemoglobin, acute chest syndrome, and hypertransfusions. Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . . This was ruled out as the MRI findings were not consistent with a specific vascular territory and normal arterial and venous flows were shown on vascular imaging. Another differential is posterior reversible encephalopathy syndrome which has been reported in SCD patients . . . . . However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury. However, it is associated to high ferritin and low triglycerides at the time of the encephalopathy, other multisystemic injuries, typical neuropathological findings, and recurrence over time, which were not noted in our patient . . Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia. It caused punctate areas of hemorrhages in the basal ganglia, periventricular white matter, and mainly along the posterior parietal cortex. This was attributed to parvovirus B19-induced vasculitis . . In our patient, there was no sign of aplasia or any neuroradiological finding of parvovirus B19 infection. Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . . This was ruled out as the patient did not have clinical or radiological signs of acute chest syndrome or embolism and there was no arterial hypoxemia. Acute hemorrhagic encephalomyelitis has been described in pediatric patients following ADEM or ADEM-like episodes . . AHEM is the most plausible diagnosis in our patients based on the clinical and radiological presentation, the preceding ADEM-like episode, and the exclusion of other etiologies of acute encephalopathy. Other patients with AHEM have been described in the SCD context . . Many treatment options have been used to treat AHEM; of these, IV steroids have been associated with survival following aggressive, high-dose corticosteroid therapy . . . . . . . . . . Autosomal dominant mutations with incomplete penetrance in RANBP2 have been associated with susceptibility to infectioninduced necrotizing encephalopathy . . Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . . Our patient has a similar presentation and imaging features as infection-induced necrotizing encephalopathy, including bilateral thalamic involvement. The rare heterozygous previously unreported variant we identified in RANBP2 affects a very conserved aminoacid and is predicted deleterious using in silico tools a prediction tool performing a fast bioinformatics analysis which can predict the pathogenicity of a variant based on the change to an amino acid . It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy . making possible that both entities might be part of the same pathophysiological continuum. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells . . Hence, RANBP2 dysfunction might make neuronal cells much vulnerable to energy failure and necrosis when exposed to inflammatory or other stresses, such as those implicated in AHEM. This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication. All authors participated in gathering the data, designing the article, and discussing and editing the manuscript. aCKNoWleDgMeNts We thank Dr. S. Abish, Dr. N. Ahmed, and Mrs. C. Guiraut for their help. We are grateful to the Hoppenheim Fund from the Montreal Children Hospital Foundation. The first author of this article received a scholarship from the Hoppenheim Fund, Montreal Children Hospital Foundation .. This work was supported by grants from Heart and Stroke Foundation of Canada grant number: G-14-0005756 , and Foundation of Stars.
What is RANBP2?	nuclear pore protein	[ "BACKGROUND: Acute hemorrhagic encephalomyelitis AHEM is considered as a rare form of acute disseminated encephalomyelitis characterized by fulminant encephalopathy with hemorrhagic necrosis and most often fatal outcome. OBJECTIVE: To report the association with Ran Binding Protein RANBP2 gene variant and the response to decompressive craniectomy and high-dose intravenous methylprednisolone IVMP in life-threatening AHEM. DESIGN: Single case study. CASE REPORT: A 6-year-old girl known to have sickle cell disease SCD presented an acquired demyelinating syndrome ADS with diplopia due to sudden unilateral fourth nerve palsy. She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma.", "She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma. Brain magnetic resonance imaging showed a left frontal necrotico-hemorrhagic lesion and new multifocal areas of demyelination. She underwent decompressive craniotomy and evacuation of an ongoing left frontoparietal hemorrhage. Comprehensive investigations ruled out vascular and infectious process. The neurological deterioration stopped concomitantly with combined neurosurgical drainage of the hematoma, decompressive craniotomy, IVMP, and intravenous immunoglobulins IVIG . She developed during the following months Crohn disease and sclerosing cholangitis. After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered.", "After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered. A heterozygous novel rare missense variant c.4993A>G, p.Lys1665Glu was identified in RANBP2, a gene associated with acute necrotizing encephalopathy. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP.", "RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP. This case report, in addition to others, stresses the likely efficacy of combined craniotomy, IVIG, and IVMP treatments in AHEM. RANBP2 mutations may sensitize the brain to inflammation and predispose to AHEM. Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system.", "Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system. It is usually fatal . . . . Many treatment options have been used including intravenous IV steroids, intravenous immunoglobulins IVIG , and plasmapheresis . . There have been few reports of survival following early intervention with high-dose corticosteroid therapy and/or decompressive craniotomy . . . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle.", ". . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle. RANBP2 is associated with microtubules and mitochondria suggesting roles in intracellular protein trafficking or energy maintenance and homeostasis of neuronal cells. RANBP2 mutations have been reported in acute necrotizing encephalopathy ANE which could present with coma, convulsions, and encephalopathy. The hallmark of ANE is multiple, symmetric brain lesions located in the thalami bilaterally, putamina, deep periventricular white matter, cerebellum, and brainstem. It could be triggered by a viral infection in previously healthy children . .", "It could be triggered by a viral infection in previously healthy children . . We report a new case of AHEM associated to a Ran Binding Protein RANBP -2 variant and responsive to combined craniectomy, intravenous methylprednisolone IVMP , and IVIG as inaugural manifestation of multisystemic autoimmunity in a girl with sickle cell disease SCD . A 6-year-old girl known for SCD treated on folic acid and hydroxyurea was admitted for new-onset diplopia day 0 D0 : refers to the start of the diplopia 6 weeks after respiratory tract infection due to rhinovirus. She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 .", "She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 . Brain MR angiography MRA showed a normal appearing circle of Willis. The cerebrospinal fluid CSF obtained by lumber puncture was normal WBC 1 cells/μl, RBC 0 cells/μl, glucose 2.9 mmol/L, protein 0.18 g/L, and absent oligoclonal bands . The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area.", "The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area. Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h.", "Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h. She was treated with five doses of IVMP 30 mg/kg/day followed by 9 days of oral prednisone 1 mg/kg/day . At discharge, her neurological exam was significant only for vertical diplopia. She presented 1 month later with 5 days of upper respiratory tract infection symptoms, fever, headache, and a rapidly progressive right-hand weakness D30 with normal alertness. She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir.", "She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir. White cell count was 13.4 × 10 9 /L, hemoglobin was 7.8 g/L, and platelets were 239 × 10 9 /L. While in the MRI machine D30 she deteriorated with vomiting and reduced level of consciousness Glasgow Coma Scale dropped from 15 to 8 over 30 min . Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F .", "Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F . The patient was immediately brought out of the magnet and her physical exam demonstrated unequal dilated pupils. She received IV mannitol and hypertonic saline for the management of acute intracranial hypertension/ herniation and was taken for surgery. She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis.", "She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis. Estimated blood loss was 3.5 L. She received 8 units of packed red blood cells, 3 units of cryoprecipitate, 6 units of fresh frozen plasma, and 3 units of platelets. Coagulation profile showed international normalization ratio = 3.38, prothrombin time = 51.2 s, and partial thromboplastin time = 122 s. An intraventricular pressure monitor was inserted. She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces.", "She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces. New white matter lesions were detected in the left posterior parietal and occipital lobes and in the left caudate head. MRI at D33 showed interval worsening with disseminated gray and white matter non-hemorrhagic lesions in the right cerebral and both cerebellar hemispheres, bilateral deep gray nuclei, as well as new necrotic non-hemorrhagic lesions in the left hemisphere Figures 2G-I . She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 .", "She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 . Prednisolone was tapered course over 6 weeks. At discharge D71 , she was able to say a few words and had better power of her right side. Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma.", "Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma. There was diffuse microglial activation and signs of early microinfarcts. Blood, CSF and urine culture, and PCR HSV1/2 were negative for bacteria and for viruses. CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative.", "CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative. Anti-extractable nuclear antigens SSA-RO, SSB-LA, smith, RNP were negative. Serum autoimmune antibodies panel NMO, NMDAR, AMPA I/II, GAB, MAG, VGCC, MOG, YO, HU, RI were negative but GAD antibody was slightly positive, possibly due to the IVIG infusion. EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange.", "EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange. Six months later, the patient was diagnosed to have Crohn's disease and primary sclerosing cholangitis. Two years later, the patient still suffers right hemiparesis but is able to walk without support. She presents an expressive aphasia. Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu .", "Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu . This mutation has not been previously reported in the HGMD database. This variant has been observed at a frequency of <0.01% across the entire Broad ExAC dataset of individuals without severe childhood onset disease 6/117,118 alleles . Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD .", "Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD . Several differential diagnoses of acute encephalopathy in a patient with sickle cell anemia can be considered. An infectious encephalitis, including herpes encephalitis, was ruled out by blood and CSF bacterial and viral cultures and negative HSV I/ II PCR. Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . .", "Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . . SCD patients are prone to ischemic or hemorrhagic strokes . . Primary hemorrhagic stroke is uncommon in pediatric SCD. Most cases were from adults and have been described in the context of previous ischemic stroke, aneurysms, low hemoglobin, acute chest syndrome, and hypertransfusions. Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . .", "Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . . This was ruled out as the MRI findings were not consistent with a specific vascular territory and normal arterial and venous flows were shown on vascular imaging. Another differential is posterior reversible encephalopathy syndrome which has been reported in SCD patients . . . . . However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury.", "However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury. However, it is associated to high ferritin and low triglycerides at the time of the encephalopathy, other multisystemic injuries, typical neuropathological findings, and recurrence over time, which were not noted in our patient . . Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia.", ". Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia. It caused punctate areas of hemorrhages in the basal ganglia, periventricular white matter, and mainly along the posterior parietal cortex. This was attributed to parvovirus B19-induced vasculitis . . In our patient, there was no sign of aplasia or any neuroradiological finding of parvovirus B19 infection. Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . .", "Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . . This was ruled out as the patient did not have clinical or radiological signs of acute chest syndrome or embolism and there was no arterial hypoxemia. Acute hemorrhagic encephalomyelitis has been described in pediatric patients following ADEM or ADEM-like episodes . . AHEM is the most plausible diagnosis in our patients based on the clinical and radiological presentation, the preceding ADEM-like episode, and the exclusion of other etiologies of acute encephalopathy. Other patients with AHEM have been described in the SCD context . .", "Other patients with AHEM have been described in the SCD context . . Many treatment options have been used to treat AHEM; of these, IV steroids have been associated with survival following aggressive, high-dose corticosteroid therapy . . . . . . . . . . Autosomal dominant mutations with incomplete penetrance in RANBP2 have been associated with susceptibility to infectioninduced necrotizing encephalopathy . . Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . .", "Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . . Our patient has a similar presentation and imaging features as infection-induced necrotizing encephalopathy, including bilateral thalamic involvement. The rare heterozygous previously unreported variant we identified in RANBP2 affects a very conserved aminoacid and is predicted deleterious using in silico tools a prediction tool performing a fast bioinformatics analysis which can predict the pathogenicity of a variant based on the change to an amino acid . It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy .", "It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy . making possible that both entities might be part of the same pathophysiological continuum. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells . . Hence, RANBP2 dysfunction might make neuronal cells much vulnerable to energy failure and necrosis when exposed to inflammatory or other stresses, such as those implicated in AHEM. This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication.", "This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication. All authors participated in gathering the data, designing the article, and discussing and editing the manuscript. aCKNoWleDgMeNts We thank Dr. S. Abish, Dr. N. Ahmed, and Mrs. C. Guiraut for their help. We are grateful to the Hoppenheim Fund from the Montreal Children Hospital Foundation. The first author of this article received a scholarship from the Hoppenheim Fund, Montreal Children Hospital Foundation .. This work was supported by grants from Heart and Stroke Foundation of Canada grant number: G-14-0005756 , and Foundation of Stars." ]	1,561	3,034	BACKGROUND: Acute hemorrhagic encephalomyelitis AHEM is considered as a rare form of acute disseminated encephalomyelitis characterized by fulminant encephalopathy with hemorrhagic necrosis and most often fatal outcome. OBJECTIVE: To report the association with Ran Binding Protein RANBP2 gene variant and the response to decompressive craniectomy and high-dose intravenous methylprednisolone IVMP in life-threatening AHEM. DESIGN: Single case study. CASE REPORT: A 6-year-old girl known to have sickle cell disease SCD presented an acquired demyelinating syndrome ADS with diplopia due to sudden unilateral fourth nerve palsy. She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma. Brain magnetic resonance imaging showed a left frontal necrotico-hemorrhagic lesion and new multifocal areas of demyelination. She underwent decompressive craniotomy and evacuation of an ongoing left frontoparietal hemorrhage. Comprehensive investigations ruled out vascular and infectious process. The neurological deterioration stopped concomitantly with combined neurosurgical drainage of the hematoma, decompressive craniotomy, IVMP, and intravenous immunoglobulins IVIG . She developed during the following months Crohn disease and sclerosing cholangitis. After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered. A heterozygous novel rare missense variant c.4993A>G, p.Lys1665Glu was identified in RANBP2, a gene associated with acute necrotizing encephalopathy. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP. This case report, in addition to others, stresses the likely efficacy of combined craniotomy, IVIG, and IVMP treatments in AHEM. RANBP2 mutations may sensitize the brain to inflammation and predispose to AHEM. Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system. It is usually fatal . . . . Many treatment options have been used including intravenous IV steroids, intravenous immunoglobulins IVIG , and plasmapheresis . . There have been few reports of survival following early intervention with high-dose corticosteroid therapy and/or decompressive craniotomy . . . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle. RANBP2 is associated with microtubules and mitochondria suggesting roles in intracellular protein trafficking or energy maintenance and homeostasis of neuronal cells. RANBP2 mutations have been reported in acute necrotizing encephalopathy ANE which could present with coma, convulsions, and encephalopathy. The hallmark of ANE is multiple, symmetric brain lesions located in the thalami bilaterally, putamina, deep periventricular white matter, cerebellum, and brainstem. It could be triggered by a viral infection in previously healthy children . . We report a new case of AHEM associated to a Ran Binding Protein RANBP -2 variant and responsive to combined craniectomy, intravenous methylprednisolone IVMP , and IVIG as inaugural manifestation of multisystemic autoimmunity in a girl with sickle cell disease SCD . A 6-year-old girl known for SCD treated on folic acid and hydroxyurea was admitted for new-onset diplopia day 0 D0 : refers to the start of the diplopia 6 weeks after respiratory tract infection due to rhinovirus. She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 . Brain MR angiography MRA showed a normal appearing circle of Willis. The cerebrospinal fluid CSF obtained by lumber puncture was normal WBC 1 cells/μl, RBC 0 cells/μl, glucose 2.9 mmol/L, protein 0.18 g/L, and absent oligoclonal bands . The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area. Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h. She was treated with five doses of IVMP 30 mg/kg/day followed by 9 days of oral prednisone 1 mg/kg/day . At discharge, her neurological exam was significant only for vertical diplopia. She presented 1 month later with 5 days of upper respiratory tract infection symptoms, fever, headache, and a rapidly progressive right-hand weakness D30 with normal alertness. She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir. White cell count was 13.4 × 10 9 /L, hemoglobin was 7.8 g/L, and platelets were 239 × 10 9 /L. While in the MRI machine D30 she deteriorated with vomiting and reduced level of consciousness Glasgow Coma Scale dropped from 15 to 8 over 30 min . Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F . The patient was immediately brought out of the magnet and her physical exam demonstrated unequal dilated pupils. She received IV mannitol and hypertonic saline for the management of acute intracranial hypertension/ herniation and was taken for surgery. She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis. Estimated blood loss was 3.5 L. She received 8 units of packed red blood cells, 3 units of cryoprecipitate, 6 units of fresh frozen plasma, and 3 units of platelets. Coagulation profile showed international normalization ratio = 3.38, prothrombin time = 51.2 s, and partial thromboplastin time = 122 s. An intraventricular pressure monitor was inserted. She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces. New white matter lesions were detected in the left posterior parietal and occipital lobes and in the left caudate head. MRI at D33 showed interval worsening with disseminated gray and white matter non-hemorrhagic lesions in the right cerebral and both cerebellar hemispheres, bilateral deep gray nuclei, as well as new necrotic non-hemorrhagic lesions in the left hemisphere Figures 2G-I . She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 . Prednisolone was tapered course over 6 weeks. At discharge D71 , she was able to say a few words and had better power of her right side. Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma. There was diffuse microglial activation and signs of early microinfarcts. Blood, CSF and urine culture, and PCR HSV1/2 were negative for bacteria and for viruses. CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative. Anti-extractable nuclear antigens SSA-RO, SSB-LA, smith, RNP were negative. Serum autoimmune antibodies panel NMO, NMDAR, AMPA I/II, GAB, MAG, VGCC, MOG, YO, HU, RI were negative but GAD antibody was slightly positive, possibly due to the IVIG infusion. EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange. Six months later, the patient was diagnosed to have Crohn's disease and primary sclerosing cholangitis. Two years later, the patient still suffers right hemiparesis but is able to walk without support. She presents an expressive aphasia. Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu . This mutation has not been previously reported in the HGMD database. This variant has been observed at a frequency of <0.01% across the entire Broad ExAC dataset of individuals without severe childhood onset disease 6/117,118 alleles . Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD . Several differential diagnoses of acute encephalopathy in a patient with sickle cell anemia can be considered. An infectious encephalitis, including herpes encephalitis, was ruled out by blood and CSF bacterial and viral cultures and negative HSV I/ II PCR. Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . . SCD patients are prone to ischemic or hemorrhagic strokes . . Primary hemorrhagic stroke is uncommon in pediatric SCD. Most cases were from adults and have been described in the context of previous ischemic stroke, aneurysms, low hemoglobin, acute chest syndrome, and hypertransfusions. Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . . This was ruled out as the MRI findings were not consistent with a specific vascular territory and normal arterial and venous flows were shown on vascular imaging. Another differential is posterior reversible encephalopathy syndrome which has been reported in SCD patients . . . . . However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury. However, it is associated to high ferritin and low triglycerides at the time of the encephalopathy, other multisystemic injuries, typical neuropathological findings, and recurrence over time, which were not noted in our patient . . Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia. It caused punctate areas of hemorrhages in the basal ganglia, periventricular white matter, and mainly along the posterior parietal cortex. This was attributed to parvovirus B19-induced vasculitis . . In our patient, there was no sign of aplasia or any neuroradiological finding of parvovirus B19 infection. Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . . This was ruled out as the patient did not have clinical or radiological signs of acute chest syndrome or embolism and there was no arterial hypoxemia. Acute hemorrhagic encephalomyelitis has been described in pediatric patients following ADEM or ADEM-like episodes . . AHEM is the most plausible diagnosis in our patients based on the clinical and radiological presentation, the preceding ADEM-like episode, and the exclusion of other etiologies of acute encephalopathy. Other patients with AHEM have been described in the SCD context . . Many treatment options have been used to treat AHEM; of these, IV steroids have been associated with survival following aggressive, high-dose corticosteroid therapy . . . . . . . . . . Autosomal dominant mutations with incomplete penetrance in RANBP2 have been associated with susceptibility to infectioninduced necrotizing encephalopathy . . Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . . Our patient has a similar presentation and imaging features as infection-induced necrotizing encephalopathy, including bilateral thalamic involvement. The rare heterozygous previously unreported variant we identified in RANBP2 affects a very conserved aminoacid and is predicted deleterious using in silico tools a prediction tool performing a fast bioinformatics analysis which can predict the pathogenicity of a variant based on the change to an amino acid . It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy . making possible that both entities might be part of the same pathophysiological continuum. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells . . Hence, RANBP2 dysfunction might make neuronal cells much vulnerable to energy failure and necrosis when exposed to inflammatory or other stresses, such as those implicated in AHEM. This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication. All authors participated in gathering the data, designing the article, and discussing and editing the manuscript. aCKNoWleDgMeNts We thank Dr. S. Abish, Dr. N. Ahmed, and Mrs. C. Guiraut for their help. We are grateful to the Hoppenheim Fund from the Montreal Children Hospital Foundation. The first author of this article received a scholarship from the Hoppenheim Fund, Montreal Children Hospital Foundation .. This work was supported by grants from Heart and Stroke Foundation of Canada grant number: G-14-0005756 , and Foundation of Stars.
What is the suggested role of RANBP2 in the cell?	intracellular protein trafficking or energy maintenance and homeostasis of neuronal cells	[ "BACKGROUND: Acute hemorrhagic encephalomyelitis AHEM is considered as a rare form of acute disseminated encephalomyelitis characterized by fulminant encephalopathy with hemorrhagic necrosis and most often fatal outcome. OBJECTIVE: To report the association with Ran Binding Protein RANBP2 gene variant and the response to decompressive craniectomy and high-dose intravenous methylprednisolone IVMP in life-threatening AHEM. DESIGN: Single case study. CASE REPORT: A 6-year-old girl known to have sickle cell disease SCD presented an acquired demyelinating syndrome ADS with diplopia due to sudden unilateral fourth nerve palsy. She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma.", "She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma. Brain magnetic resonance imaging showed a left frontal necrotico-hemorrhagic lesion and new multifocal areas of demyelination. She underwent decompressive craniotomy and evacuation of an ongoing left frontoparietal hemorrhage. Comprehensive investigations ruled out vascular and infectious process. The neurological deterioration stopped concomitantly with combined neurosurgical drainage of the hematoma, decompressive craniotomy, IVMP, and intravenous immunoglobulins IVIG . She developed during the following months Crohn disease and sclerosing cholangitis. After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered.", "After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered. A heterozygous novel rare missense variant c.4993A>G, p.Lys1665Glu was identified in RANBP2, a gene associated with acute necrotizing encephalopathy. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP.", "RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP. This case report, in addition to others, stresses the likely efficacy of combined craniotomy, IVIG, and IVMP treatments in AHEM. RANBP2 mutations may sensitize the brain to inflammation and predispose to AHEM. Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system.", "Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system. It is usually fatal . . . . Many treatment options have been used including intravenous IV steroids, intravenous immunoglobulins IVIG , and plasmapheresis . . There have been few reports of survival following early intervention with high-dose corticosteroid therapy and/or decompressive craniotomy . . . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle.", ". . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle. RANBP2 is associated with microtubules and mitochondria suggesting roles in intracellular protein trafficking or energy maintenance and homeostasis of neuronal cells. RANBP2 mutations have been reported in acute necrotizing encephalopathy ANE which could present with coma, convulsions, and encephalopathy. The hallmark of ANE is multiple, symmetric brain lesions located in the thalami bilaterally, putamina, deep periventricular white matter, cerebellum, and brainstem. It could be triggered by a viral infection in previously healthy children . .", "It could be triggered by a viral infection in previously healthy children . . We report a new case of AHEM associated to a Ran Binding Protein RANBP -2 variant and responsive to combined craniectomy, intravenous methylprednisolone IVMP , and IVIG as inaugural manifestation of multisystemic autoimmunity in a girl with sickle cell disease SCD . A 6-year-old girl known for SCD treated on folic acid and hydroxyurea was admitted for new-onset diplopia day 0 D0 : refers to the start of the diplopia 6 weeks after respiratory tract infection due to rhinovirus. She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 .", "She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 . Brain MR angiography MRA showed a normal appearing circle of Willis. The cerebrospinal fluid CSF obtained by lumber puncture was normal WBC 1 cells/μl, RBC 0 cells/μl, glucose 2.9 mmol/L, protein 0.18 g/L, and absent oligoclonal bands . The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area.", "The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area. Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h.", "Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h. She was treated with five doses of IVMP 30 mg/kg/day followed by 9 days of oral prednisone 1 mg/kg/day . At discharge, her neurological exam was significant only for vertical diplopia. She presented 1 month later with 5 days of upper respiratory tract infection symptoms, fever, headache, and a rapidly progressive right-hand weakness D30 with normal alertness. She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir.", "She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir. White cell count was 13.4 × 10 9 /L, hemoglobin was 7.8 g/L, and platelets were 239 × 10 9 /L. While in the MRI machine D30 she deteriorated with vomiting and reduced level of consciousness Glasgow Coma Scale dropped from 15 to 8 over 30 min . Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F .", "Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F . The patient was immediately brought out of the magnet and her physical exam demonstrated unequal dilated pupils. She received IV mannitol and hypertonic saline for the management of acute intracranial hypertension/ herniation and was taken for surgery. She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis.", "She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis. Estimated blood loss was 3.5 L. She received 8 units of packed red blood cells, 3 units of cryoprecipitate, 6 units of fresh frozen plasma, and 3 units of platelets. Coagulation profile showed international normalization ratio = 3.38, prothrombin time = 51.2 s, and partial thromboplastin time = 122 s. An intraventricular pressure monitor was inserted. She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces.", "She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces. New white matter lesions were detected in the left posterior parietal and occipital lobes and in the left caudate head. MRI at D33 showed interval worsening with disseminated gray and white matter non-hemorrhagic lesions in the right cerebral and both cerebellar hemispheres, bilateral deep gray nuclei, as well as new necrotic non-hemorrhagic lesions in the left hemisphere Figures 2G-I . She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 .", "She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 . Prednisolone was tapered course over 6 weeks. At discharge D71 , she was able to say a few words and had better power of her right side. Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma.", "Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma. There was diffuse microglial activation and signs of early microinfarcts. Blood, CSF and urine culture, and PCR HSV1/2 were negative for bacteria and for viruses. CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative.", "CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative. Anti-extractable nuclear antigens SSA-RO, SSB-LA, smith, RNP were negative. Serum autoimmune antibodies panel NMO, NMDAR, AMPA I/II, GAB, MAG, VGCC, MOG, YO, HU, RI were negative but GAD antibody was slightly positive, possibly due to the IVIG infusion. EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange.", "EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange. Six months later, the patient was diagnosed to have Crohn's disease and primary sclerosing cholangitis. Two years later, the patient still suffers right hemiparesis but is able to walk without support. She presents an expressive aphasia. Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu .", "Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu . This mutation has not been previously reported in the HGMD database. This variant has been observed at a frequency of <0.01% across the entire Broad ExAC dataset of individuals without severe childhood onset disease 6/117,118 alleles . Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD .", "Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD . Several differential diagnoses of acute encephalopathy in a patient with sickle cell anemia can be considered. An infectious encephalitis, including herpes encephalitis, was ruled out by blood and CSF bacterial and viral cultures and negative HSV I/ II PCR. Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . .", "Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . . SCD patients are prone to ischemic or hemorrhagic strokes . . Primary hemorrhagic stroke is uncommon in pediatric SCD. Most cases were from adults and have been described in the context of previous ischemic stroke, aneurysms, low hemoglobin, acute chest syndrome, and hypertransfusions. Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . .", "Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . . This was ruled out as the MRI findings were not consistent with a specific vascular territory and normal arterial and venous flows were shown on vascular imaging. Another differential is posterior reversible encephalopathy syndrome which has been reported in SCD patients . . . . . However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury.", "However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury. However, it is associated to high ferritin and low triglycerides at the time of the encephalopathy, other multisystemic injuries, typical neuropathological findings, and recurrence over time, which were not noted in our patient . . Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia.", ". Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia. It caused punctate areas of hemorrhages in the basal ganglia, periventricular white matter, and mainly along the posterior parietal cortex. This was attributed to parvovirus B19-induced vasculitis . . In our patient, there was no sign of aplasia or any neuroradiological finding of parvovirus B19 infection. Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . .", "Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . . This was ruled out as the patient did not have clinical or radiological signs of acute chest syndrome or embolism and there was no arterial hypoxemia. Acute hemorrhagic encephalomyelitis has been described in pediatric patients following ADEM or ADEM-like episodes . . AHEM is the most plausible diagnosis in our patients based on the clinical and radiological presentation, the preceding ADEM-like episode, and the exclusion of other etiologies of acute encephalopathy. Other patients with AHEM have been described in the SCD context . .", "Other patients with AHEM have been described in the SCD context . . Many treatment options have been used to treat AHEM; of these, IV steroids have been associated with survival following aggressive, high-dose corticosteroid therapy . . . . . . . . . . Autosomal dominant mutations with incomplete penetrance in RANBP2 have been associated with susceptibility to infectioninduced necrotizing encephalopathy . . Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . .", "Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . . Our patient has a similar presentation and imaging features as infection-induced necrotizing encephalopathy, including bilateral thalamic involvement. The rare heterozygous previously unreported variant we identified in RANBP2 affects a very conserved aminoacid and is predicted deleterious using in silico tools a prediction tool performing a fast bioinformatics analysis which can predict the pathogenicity of a variant based on the change to an amino acid . It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy .", "It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy . making possible that both entities might be part of the same pathophysiological continuum. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells . . Hence, RANBP2 dysfunction might make neuronal cells much vulnerable to energy failure and necrosis when exposed to inflammatory or other stresses, such as those implicated in AHEM. This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication.", "This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication. All authors participated in gathering the data, designing the article, and discussing and editing the manuscript. aCKNoWleDgMeNts We thank Dr. S. Abish, Dr. N. Ahmed, and Mrs. C. Guiraut for their help. We are grateful to the Hoppenheim Fund from the Montreal Children Hospital Foundation. The first author of this article received a scholarship from the Hoppenheim Fund, Montreal Children Hospital Foundation .. This work was supported by grants from Heart and Stroke Foundation of Canada grant number: G-14-0005756 , and Foundation of Stars." ]	1,561	3,035	BACKGROUND: Acute hemorrhagic encephalomyelitis AHEM is considered as a rare form of acute disseminated encephalomyelitis characterized by fulminant encephalopathy with hemorrhagic necrosis and most often fatal outcome. OBJECTIVE: To report the association with Ran Binding Protein RANBP2 gene variant and the response to decompressive craniectomy and high-dose intravenous methylprednisolone IVMP in life-threatening AHEM. DESIGN: Single case study. CASE REPORT: A 6-year-old girl known to have sickle cell disease SCD presented an acquired demyelinating syndrome ADS with diplopia due to sudden unilateral fourth nerve palsy. She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma. Brain magnetic resonance imaging showed a left frontal necrotico-hemorrhagic lesion and new multifocal areas of demyelination. She underwent decompressive craniotomy and evacuation of an ongoing left frontoparietal hemorrhage. Comprehensive investigations ruled out vascular and infectious process. The neurological deterioration stopped concomitantly with combined neurosurgical drainage of the hematoma, decompressive craniotomy, IVMP, and intravenous immunoglobulins IVIG . She developed during the following months Crohn disease and sclerosing cholangitis. After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered. A heterozygous novel rare missense variant c.4993A>G, p.Lys1665Glu was identified in RANBP2, a gene associated with acute necrotizing encephalopathy. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP. This case report, in addition to others, stresses the likely efficacy of combined craniotomy, IVIG, and IVMP treatments in AHEM. RANBP2 mutations may sensitize the brain to inflammation and predispose to AHEM. Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system. It is usually fatal . . . . Many treatment options have been used including intravenous IV steroids, intravenous immunoglobulins IVIG , and plasmapheresis . . There have been few reports of survival following early intervention with high-dose corticosteroid therapy and/or decompressive craniotomy . . . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle. RANBP2 is associated with microtubules and mitochondria suggesting roles in intracellular protein trafficking or energy maintenance and homeostasis of neuronal cells. RANBP2 mutations have been reported in acute necrotizing encephalopathy ANE which could present with coma, convulsions, and encephalopathy. The hallmark of ANE is multiple, symmetric brain lesions located in the thalami bilaterally, putamina, deep periventricular white matter, cerebellum, and brainstem. It could be triggered by a viral infection in previously healthy children . . We report a new case of AHEM associated to a Ran Binding Protein RANBP -2 variant and responsive to combined craniectomy, intravenous methylprednisolone IVMP , and IVIG as inaugural manifestation of multisystemic autoimmunity in a girl with sickle cell disease SCD . A 6-year-old girl known for SCD treated on folic acid and hydroxyurea was admitted for new-onset diplopia day 0 D0 : refers to the start of the diplopia 6 weeks after respiratory tract infection due to rhinovirus. She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 . Brain MR angiography MRA showed a normal appearing circle of Willis. The cerebrospinal fluid CSF obtained by lumber puncture was normal WBC 1 cells/μl, RBC 0 cells/μl, glucose 2.9 mmol/L, protein 0.18 g/L, and absent oligoclonal bands . The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area. Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h. She was treated with five doses of IVMP 30 mg/kg/day followed by 9 days of oral prednisone 1 mg/kg/day . At discharge, her neurological exam was significant only for vertical diplopia. She presented 1 month later with 5 days of upper respiratory tract infection symptoms, fever, headache, and a rapidly progressive right-hand weakness D30 with normal alertness. She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir. White cell count was 13.4 × 10 9 /L, hemoglobin was 7.8 g/L, and platelets were 239 × 10 9 /L. While in the MRI machine D30 she deteriorated with vomiting and reduced level of consciousness Glasgow Coma Scale dropped from 15 to 8 over 30 min . Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F . The patient was immediately brought out of the magnet and her physical exam demonstrated unequal dilated pupils. She received IV mannitol and hypertonic saline for the management of acute intracranial hypertension/ herniation and was taken for surgery. She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis. Estimated blood loss was 3.5 L. She received 8 units of packed red blood cells, 3 units of cryoprecipitate, 6 units of fresh frozen plasma, and 3 units of platelets. Coagulation profile showed international normalization ratio = 3.38, prothrombin time = 51.2 s, and partial thromboplastin time = 122 s. An intraventricular pressure monitor was inserted. She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces. New white matter lesions were detected in the left posterior parietal and occipital lobes and in the left caudate head. MRI at D33 showed interval worsening with disseminated gray and white matter non-hemorrhagic lesions in the right cerebral and both cerebellar hemispheres, bilateral deep gray nuclei, as well as new necrotic non-hemorrhagic lesions in the left hemisphere Figures 2G-I . She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 . Prednisolone was tapered course over 6 weeks. At discharge D71 , she was able to say a few words and had better power of her right side. Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma. There was diffuse microglial activation and signs of early microinfarcts. Blood, CSF and urine culture, and PCR HSV1/2 were negative for bacteria and for viruses. CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative. Anti-extractable nuclear antigens SSA-RO, SSB-LA, smith, RNP were negative. Serum autoimmune antibodies panel NMO, NMDAR, AMPA I/II, GAB, MAG, VGCC, MOG, YO, HU, RI were negative but GAD antibody was slightly positive, possibly due to the IVIG infusion. EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange. Six months later, the patient was diagnosed to have Crohn's disease and primary sclerosing cholangitis. Two years later, the patient still suffers right hemiparesis but is able to walk without support. She presents an expressive aphasia. Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu . This mutation has not been previously reported in the HGMD database. This variant has been observed at a frequency of <0.01% across the entire Broad ExAC dataset of individuals without severe childhood onset disease 6/117,118 alleles . Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD . Several differential diagnoses of acute encephalopathy in a patient with sickle cell anemia can be considered. An infectious encephalitis, including herpes encephalitis, was ruled out by blood and CSF bacterial and viral cultures and negative HSV I/ II PCR. Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . . SCD patients are prone to ischemic or hemorrhagic strokes . . Primary hemorrhagic stroke is uncommon in pediatric SCD. Most cases were from adults and have been described in the context of previous ischemic stroke, aneurysms, low hemoglobin, acute chest syndrome, and hypertransfusions. Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . . This was ruled out as the MRI findings were not consistent with a specific vascular territory and normal arterial and venous flows were shown on vascular imaging. Another differential is posterior reversible encephalopathy syndrome which has been reported in SCD patients . . . . . However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury. However, it is associated to high ferritin and low triglycerides at the time of the encephalopathy, other multisystemic injuries, typical neuropathological findings, and recurrence over time, which were not noted in our patient . . Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia. It caused punctate areas of hemorrhages in the basal ganglia, periventricular white matter, and mainly along the posterior parietal cortex. This was attributed to parvovirus B19-induced vasculitis . . In our patient, there was no sign of aplasia or any neuroradiological finding of parvovirus B19 infection. Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . . This was ruled out as the patient did not have clinical or radiological signs of acute chest syndrome or embolism and there was no arterial hypoxemia. Acute hemorrhagic encephalomyelitis has been described in pediatric patients following ADEM or ADEM-like episodes . . AHEM is the most plausible diagnosis in our patients based on the clinical and radiological presentation, the preceding ADEM-like episode, and the exclusion of other etiologies of acute encephalopathy. Other patients with AHEM have been described in the SCD context . . Many treatment options have been used to treat AHEM; of these, IV steroids have been associated with survival following aggressive, high-dose corticosteroid therapy . . . . . . . . . . Autosomal dominant mutations with incomplete penetrance in RANBP2 have been associated with susceptibility to infectioninduced necrotizing encephalopathy . . Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . . Our patient has a similar presentation and imaging features as infection-induced necrotizing encephalopathy, including bilateral thalamic involvement. The rare heterozygous previously unreported variant we identified in RANBP2 affects a very conserved aminoacid and is predicted deleterious using in silico tools a prediction tool performing a fast bioinformatics analysis which can predict the pathogenicity of a variant based on the change to an amino acid . It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy . making possible that both entities might be part of the same pathophysiological continuum. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells . . Hence, RANBP2 dysfunction might make neuronal cells much vulnerable to energy failure and necrosis when exposed to inflammatory or other stresses, such as those implicated in AHEM. This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication. All authors participated in gathering the data, designing the article, and discussing and editing the manuscript. aCKNoWleDgMeNts We thank Dr. S. Abish, Dr. N. Ahmed, and Mrs. C. Guiraut for their help. We are grateful to the Hoppenheim Fund from the Montreal Children Hospital Foundation. The first author of this article received a scholarship from the Hoppenheim Fund, Montreal Children Hospital Foundation .. This work was supported by grants from Heart and Stroke Foundation of Canada grant number: G-14-0005756 , and Foundation of Stars.
What is the hallmark finding of acute necrotizing encephalopathy?	multiple, symmetric brain lesions located in the thalami bilaterally, putamina, deep periventricular white matter, cerebellum, and brainstem	[ "BACKGROUND: Acute hemorrhagic encephalomyelitis AHEM is considered as a rare form of acute disseminated encephalomyelitis characterized by fulminant encephalopathy with hemorrhagic necrosis and most often fatal outcome. OBJECTIVE: To report the association with Ran Binding Protein RANBP2 gene variant and the response to decompressive craniectomy and high-dose intravenous methylprednisolone IVMP in life-threatening AHEM. DESIGN: Single case study. CASE REPORT: A 6-year-old girl known to have sickle cell disease SCD presented an acquired demyelinating syndrome ADS with diplopia due to sudden unilateral fourth nerve palsy. She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma.", "She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma. Brain magnetic resonance imaging showed a left frontal necrotico-hemorrhagic lesion and new multifocal areas of demyelination. She underwent decompressive craniotomy and evacuation of an ongoing left frontoparietal hemorrhage. Comprehensive investigations ruled out vascular and infectious process. The neurological deterioration stopped concomitantly with combined neurosurgical drainage of the hematoma, decompressive craniotomy, IVMP, and intravenous immunoglobulins IVIG . She developed during the following months Crohn disease and sclerosing cholangitis. After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered.", "After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered. A heterozygous novel rare missense variant c.4993A>G, p.Lys1665Glu was identified in RANBP2, a gene associated with acute necrotizing encephalopathy. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP.", "RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP. This case report, in addition to others, stresses the likely efficacy of combined craniotomy, IVIG, and IVMP treatments in AHEM. RANBP2 mutations may sensitize the brain to inflammation and predispose to AHEM. Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system.", "Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system. It is usually fatal . . . . Many treatment options have been used including intravenous IV steroids, intravenous immunoglobulins IVIG , and plasmapheresis . . There have been few reports of survival following early intervention with high-dose corticosteroid therapy and/or decompressive craniotomy . . . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle.", ". . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle. RANBP2 is associated with microtubules and mitochondria suggesting roles in intracellular protein trafficking or energy maintenance and homeostasis of neuronal cells. RANBP2 mutations have been reported in acute necrotizing encephalopathy ANE which could present with coma, convulsions, and encephalopathy. The hallmark of ANE is multiple, symmetric brain lesions located in the thalami bilaterally, putamina, deep periventricular white matter, cerebellum, and brainstem. It could be triggered by a viral infection in previously healthy children . .", "It could be triggered by a viral infection in previously healthy children . . We report a new case of AHEM associated to a Ran Binding Protein RANBP -2 variant and responsive to combined craniectomy, intravenous methylprednisolone IVMP , and IVIG as inaugural manifestation of multisystemic autoimmunity in a girl with sickle cell disease SCD . A 6-year-old girl known for SCD treated on folic acid and hydroxyurea was admitted for new-onset diplopia day 0 D0 : refers to the start of the diplopia 6 weeks after respiratory tract infection due to rhinovirus. She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 .", "She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 . Brain MR angiography MRA showed a normal appearing circle of Willis. The cerebrospinal fluid CSF obtained by lumber puncture was normal WBC 1 cells/μl, RBC 0 cells/μl, glucose 2.9 mmol/L, protein 0.18 g/L, and absent oligoclonal bands . The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area.", "The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area. Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h.", "Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h. She was treated with five doses of IVMP 30 mg/kg/day followed by 9 days of oral prednisone 1 mg/kg/day . At discharge, her neurological exam was significant only for vertical diplopia. She presented 1 month later with 5 days of upper respiratory tract infection symptoms, fever, headache, and a rapidly progressive right-hand weakness D30 with normal alertness. She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir.", "She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir. White cell count was 13.4 × 10 9 /L, hemoglobin was 7.8 g/L, and platelets were 239 × 10 9 /L. While in the MRI machine D30 she deteriorated with vomiting and reduced level of consciousness Glasgow Coma Scale dropped from 15 to 8 over 30 min . Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F .", "Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F . The patient was immediately brought out of the magnet and her physical exam demonstrated unequal dilated pupils. She received IV mannitol and hypertonic saline for the management of acute intracranial hypertension/ herniation and was taken for surgery. She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis.", "She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis. Estimated blood loss was 3.5 L. She received 8 units of packed red blood cells, 3 units of cryoprecipitate, 6 units of fresh frozen plasma, and 3 units of platelets. Coagulation profile showed international normalization ratio = 3.38, prothrombin time = 51.2 s, and partial thromboplastin time = 122 s. An intraventricular pressure monitor was inserted. She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces.", "She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces. New white matter lesions were detected in the left posterior parietal and occipital lobes and in the left caudate head. MRI at D33 showed interval worsening with disseminated gray and white matter non-hemorrhagic lesions in the right cerebral and both cerebellar hemispheres, bilateral deep gray nuclei, as well as new necrotic non-hemorrhagic lesions in the left hemisphere Figures 2G-I . She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 .", "She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 . Prednisolone was tapered course over 6 weeks. At discharge D71 , she was able to say a few words and had better power of her right side. Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma.", "Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma. There was diffuse microglial activation and signs of early microinfarcts. Blood, CSF and urine culture, and PCR HSV1/2 were negative for bacteria and for viruses. CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative.", "CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative. Anti-extractable nuclear antigens SSA-RO, SSB-LA, smith, RNP were negative. Serum autoimmune antibodies panel NMO, NMDAR, AMPA I/II, GAB, MAG, VGCC, MOG, YO, HU, RI were negative but GAD antibody was slightly positive, possibly due to the IVIG infusion. EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange.", "EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange. Six months later, the patient was diagnosed to have Crohn's disease and primary sclerosing cholangitis. Two years later, the patient still suffers right hemiparesis but is able to walk without support. She presents an expressive aphasia. Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu .", "Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu . This mutation has not been previously reported in the HGMD database. This variant has been observed at a frequency of <0.01% across the entire Broad ExAC dataset of individuals without severe childhood onset disease 6/117,118 alleles . Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD .", "Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD . Several differential diagnoses of acute encephalopathy in a patient with sickle cell anemia can be considered. An infectious encephalitis, including herpes encephalitis, was ruled out by blood and CSF bacterial and viral cultures and negative HSV I/ II PCR. Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . .", "Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . . SCD patients are prone to ischemic or hemorrhagic strokes . . Primary hemorrhagic stroke is uncommon in pediatric SCD. Most cases were from adults and have been described in the context of previous ischemic stroke, aneurysms, low hemoglobin, acute chest syndrome, and hypertransfusions. Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . .", "Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . . This was ruled out as the MRI findings were not consistent with a specific vascular territory and normal arterial and venous flows were shown on vascular imaging. Another differential is posterior reversible encephalopathy syndrome which has been reported in SCD patients . . . . . However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury.", "However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury. However, it is associated to high ferritin and low triglycerides at the time of the encephalopathy, other multisystemic injuries, typical neuropathological findings, and recurrence over time, which were not noted in our patient . . Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia.", ". Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia. It caused punctate areas of hemorrhages in the basal ganglia, periventricular white matter, and mainly along the posterior parietal cortex. This was attributed to parvovirus B19-induced vasculitis . . In our patient, there was no sign of aplasia or any neuroradiological finding of parvovirus B19 infection. Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . .", "Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . . This was ruled out as the patient did not have clinical or radiological signs of acute chest syndrome or embolism and there was no arterial hypoxemia. Acute hemorrhagic encephalomyelitis has been described in pediatric patients following ADEM or ADEM-like episodes . . AHEM is the most plausible diagnosis in our patients based on the clinical and radiological presentation, the preceding ADEM-like episode, and the exclusion of other etiologies of acute encephalopathy. Other patients with AHEM have been described in the SCD context . .", "Other patients with AHEM have been described in the SCD context . . Many treatment options have been used to treat AHEM; of these, IV steroids have been associated with survival following aggressive, high-dose corticosteroid therapy . . . . . . . . . . Autosomal dominant mutations with incomplete penetrance in RANBP2 have been associated with susceptibility to infectioninduced necrotizing encephalopathy . . Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . .", "Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . . Our patient has a similar presentation and imaging features as infection-induced necrotizing encephalopathy, including bilateral thalamic involvement. The rare heterozygous previously unreported variant we identified in RANBP2 affects a very conserved aminoacid and is predicted deleterious using in silico tools a prediction tool performing a fast bioinformatics analysis which can predict the pathogenicity of a variant based on the change to an amino acid . It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy .", "It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy . making possible that both entities might be part of the same pathophysiological continuum. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells . . Hence, RANBP2 dysfunction might make neuronal cells much vulnerable to energy failure and necrosis when exposed to inflammatory or other stresses, such as those implicated in AHEM. This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication.", "This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication. All authors participated in gathering the data, designing the article, and discussing and editing the manuscript. aCKNoWleDgMeNts We thank Dr. S. Abish, Dr. N. Ahmed, and Mrs. C. Guiraut for their help. We are grateful to the Hoppenheim Fund from the Montreal Children Hospital Foundation. The first author of this article received a scholarship from the Hoppenheim Fund, Montreal Children Hospital Foundation .. This work was supported by grants from Heart and Stroke Foundation of Canada grant number: G-14-0005756 , and Foundation of Stars." ]	1,561	3,036	BACKGROUND: Acute hemorrhagic encephalomyelitis AHEM is considered as a rare form of acute disseminated encephalomyelitis characterized by fulminant encephalopathy with hemorrhagic necrosis and most often fatal outcome. OBJECTIVE: To report the association with Ran Binding Protein RANBP2 gene variant and the response to decompressive craniectomy and high-dose intravenous methylprednisolone IVMP in life-threatening AHEM. DESIGN: Single case study. CASE REPORT: A 6-year-old girl known to have sickle cell disease SCD presented an acquired demyelinating syndrome ADS with diplopia due to sudden unilateral fourth nerve palsy. She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma. Brain magnetic resonance imaging showed a left frontal necrotico-hemorrhagic lesion and new multifocal areas of demyelination. She underwent decompressive craniotomy and evacuation of an ongoing left frontoparietal hemorrhage. Comprehensive investigations ruled out vascular and infectious process. The neurological deterioration stopped concomitantly with combined neurosurgical drainage of the hematoma, decompressive craniotomy, IVMP, and intravenous immunoglobulins IVIG . She developed during the following months Crohn disease and sclerosing cholangitis. After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered. A heterozygous novel rare missense variant c.4993A>G, p.Lys1665Glu was identified in RANBP2, a gene associated with acute necrotizing encephalopathy. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP. This case report, in addition to others, stresses the likely efficacy of combined craniotomy, IVIG, and IVMP treatments in AHEM. RANBP2 mutations may sensitize the brain to inflammation and predispose to AHEM. Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system. It is usually fatal . . . . Many treatment options have been used including intravenous IV steroids, intravenous immunoglobulins IVIG , and plasmapheresis . . There have been few reports of survival following early intervention with high-dose corticosteroid therapy and/or decompressive craniotomy . . . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle. RANBP2 is associated with microtubules and mitochondria suggesting roles in intracellular protein trafficking or energy maintenance and homeostasis of neuronal cells. RANBP2 mutations have been reported in acute necrotizing encephalopathy ANE which could present with coma, convulsions, and encephalopathy. The hallmark of ANE is multiple, symmetric brain lesions located in the thalami bilaterally, putamina, deep periventricular white matter, cerebellum, and brainstem. It could be triggered by a viral infection in previously healthy children . . We report a new case of AHEM associated to a Ran Binding Protein RANBP -2 variant and responsive to combined craniectomy, intravenous methylprednisolone IVMP , and IVIG as inaugural manifestation of multisystemic autoimmunity in a girl with sickle cell disease SCD . A 6-year-old girl known for SCD treated on folic acid and hydroxyurea was admitted for new-onset diplopia day 0 D0 : refers to the start of the diplopia 6 weeks after respiratory tract infection due to rhinovirus. She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 . Brain MR angiography MRA showed a normal appearing circle of Willis. The cerebrospinal fluid CSF obtained by lumber puncture was normal WBC 1 cells/μl, RBC 0 cells/μl, glucose 2.9 mmol/L, protein 0.18 g/L, and absent oligoclonal bands . The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area. Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h. She was treated with five doses of IVMP 30 mg/kg/day followed by 9 days of oral prednisone 1 mg/kg/day . At discharge, her neurological exam was significant only for vertical diplopia. She presented 1 month later with 5 days of upper respiratory tract infection symptoms, fever, headache, and a rapidly progressive right-hand weakness D30 with normal alertness. She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir. White cell count was 13.4 × 10 9 /L, hemoglobin was 7.8 g/L, and platelets were 239 × 10 9 /L. While in the MRI machine D30 she deteriorated with vomiting and reduced level of consciousness Glasgow Coma Scale dropped from 15 to 8 over 30 min . Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F . The patient was immediately brought out of the magnet and her physical exam demonstrated unequal dilated pupils. She received IV mannitol and hypertonic saline for the management of acute intracranial hypertension/ herniation and was taken for surgery. She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis. Estimated blood loss was 3.5 L. She received 8 units of packed red blood cells, 3 units of cryoprecipitate, 6 units of fresh frozen plasma, and 3 units of platelets. Coagulation profile showed international normalization ratio = 3.38, prothrombin time = 51.2 s, and partial thromboplastin time = 122 s. An intraventricular pressure monitor was inserted. She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces. New white matter lesions were detected in the left posterior parietal and occipital lobes and in the left caudate head. MRI at D33 showed interval worsening with disseminated gray and white matter non-hemorrhagic lesions in the right cerebral and both cerebellar hemispheres, bilateral deep gray nuclei, as well as new necrotic non-hemorrhagic lesions in the left hemisphere Figures 2G-I . She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 . Prednisolone was tapered course over 6 weeks. At discharge D71 , she was able to say a few words and had better power of her right side. Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma. There was diffuse microglial activation and signs of early microinfarcts. Blood, CSF and urine culture, and PCR HSV1/2 were negative for bacteria and for viruses. CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative. Anti-extractable nuclear antigens SSA-RO, SSB-LA, smith, RNP were negative. Serum autoimmune antibodies panel NMO, NMDAR, AMPA I/II, GAB, MAG, VGCC, MOG, YO, HU, RI were negative but GAD antibody was slightly positive, possibly due to the IVIG infusion. EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange. Six months later, the patient was diagnosed to have Crohn's disease and primary sclerosing cholangitis. Two years later, the patient still suffers right hemiparesis but is able to walk without support. She presents an expressive aphasia. Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu . This mutation has not been previously reported in the HGMD database. This variant has been observed at a frequency of <0.01% across the entire Broad ExAC dataset of individuals without severe childhood onset disease 6/117,118 alleles . Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD . Several differential diagnoses of acute encephalopathy in a patient with sickle cell anemia can be considered. An infectious encephalitis, including herpes encephalitis, was ruled out by blood and CSF bacterial and viral cultures and negative HSV I/ II PCR. Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . . SCD patients are prone to ischemic or hemorrhagic strokes . . Primary hemorrhagic stroke is uncommon in pediatric SCD. Most cases were from adults and have been described in the context of previous ischemic stroke, aneurysms, low hemoglobin, acute chest syndrome, and hypertransfusions. Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . . This was ruled out as the MRI findings were not consistent with a specific vascular territory and normal arterial and venous flows were shown on vascular imaging. Another differential is posterior reversible encephalopathy syndrome which has been reported in SCD patients . . . . . However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury. However, it is associated to high ferritin and low triglycerides at the time of the encephalopathy, other multisystemic injuries, typical neuropathological findings, and recurrence over time, which were not noted in our patient . . Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia. It caused punctate areas of hemorrhages in the basal ganglia, periventricular white matter, and mainly along the posterior parietal cortex. This was attributed to parvovirus B19-induced vasculitis . . In our patient, there was no sign of aplasia or any neuroradiological finding of parvovirus B19 infection. Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . . This was ruled out as the patient did not have clinical or radiological signs of acute chest syndrome or embolism and there was no arterial hypoxemia. Acute hemorrhagic encephalomyelitis has been described in pediatric patients following ADEM or ADEM-like episodes . . AHEM is the most plausible diagnosis in our patients based on the clinical and radiological presentation, the preceding ADEM-like episode, and the exclusion of other etiologies of acute encephalopathy. Other patients with AHEM have been described in the SCD context . . Many treatment options have been used to treat AHEM; of these, IV steroids have been associated with survival following aggressive, high-dose corticosteroid therapy . . . . . . . . . . Autosomal dominant mutations with incomplete penetrance in RANBP2 have been associated with susceptibility to infectioninduced necrotizing encephalopathy . . Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . . Our patient has a similar presentation and imaging features as infection-induced necrotizing encephalopathy, including bilateral thalamic involvement. The rare heterozygous previously unreported variant we identified in RANBP2 affects a very conserved aminoacid and is predicted deleterious using in silico tools a prediction tool performing a fast bioinformatics analysis which can predict the pathogenicity of a variant based on the change to an amino acid . It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy . making possible that both entities might be part of the same pathophysiological continuum. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells . . Hence, RANBP2 dysfunction might make neuronal cells much vulnerable to energy failure and necrosis when exposed to inflammatory or other stresses, such as those implicated in AHEM. This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication. All authors participated in gathering the data, designing the article, and discussing and editing the manuscript. aCKNoWleDgMeNts We thank Dr. S. Abish, Dr. N. Ahmed, and Mrs. C. Guiraut for their help. We are grateful to the Hoppenheim Fund from the Montreal Children Hospital Foundation. The first author of this article received a scholarship from the Hoppenheim Fund, Montreal Children Hospital Foundation .. This work was supported by grants from Heart and Stroke Foundation of Canada grant number: G-14-0005756 , and Foundation of Stars.
What could trigger acute necrotizing encephalopathy?	viral infection in previously healthy children	[ "BACKGROUND: Acute hemorrhagic encephalomyelitis AHEM is considered as a rare form of acute disseminated encephalomyelitis characterized by fulminant encephalopathy with hemorrhagic necrosis and most often fatal outcome. OBJECTIVE: To report the association with Ran Binding Protein RANBP2 gene variant and the response to decompressive craniectomy and high-dose intravenous methylprednisolone IVMP in life-threatening AHEM. DESIGN: Single case study. CASE REPORT: A 6-year-old girl known to have sickle cell disease SCD presented an acquired demyelinating syndrome ADS with diplopia due to sudden unilateral fourth nerve palsy. She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma.", "She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma. Brain magnetic resonance imaging showed a left frontal necrotico-hemorrhagic lesion and new multifocal areas of demyelination. She underwent decompressive craniotomy and evacuation of an ongoing left frontoparietal hemorrhage. Comprehensive investigations ruled out vascular and infectious process. The neurological deterioration stopped concomitantly with combined neurosurgical drainage of the hematoma, decompressive craniotomy, IVMP, and intravenous immunoglobulins IVIG . She developed during the following months Crohn disease and sclerosing cholangitis. After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered.", "After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered. A heterozygous novel rare missense variant c.4993A>G, p.Lys1665Glu was identified in RANBP2, a gene associated with acute necrotizing encephalopathy. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP.", "RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP. This case report, in addition to others, stresses the likely efficacy of combined craniotomy, IVIG, and IVMP treatments in AHEM. RANBP2 mutations may sensitize the brain to inflammation and predispose to AHEM. Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system.", "Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system. It is usually fatal . . . . Many treatment options have been used including intravenous IV steroids, intravenous immunoglobulins IVIG , and plasmapheresis . . There have been few reports of survival following early intervention with high-dose corticosteroid therapy and/or decompressive craniotomy . . . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle.", ". . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle. RANBP2 is associated with microtubules and mitochondria suggesting roles in intracellular protein trafficking or energy maintenance and homeostasis of neuronal cells. RANBP2 mutations have been reported in acute necrotizing encephalopathy ANE which could present with coma, convulsions, and encephalopathy. The hallmark of ANE is multiple, symmetric brain lesions located in the thalami bilaterally, putamina, deep periventricular white matter, cerebellum, and brainstem. It could be triggered by a viral infection in previously healthy children . .", "It could be triggered by a viral infection in previously healthy children . . We report a new case of AHEM associated to a Ran Binding Protein RANBP -2 variant and responsive to combined craniectomy, intravenous methylprednisolone IVMP , and IVIG as inaugural manifestation of multisystemic autoimmunity in a girl with sickle cell disease SCD . A 6-year-old girl known for SCD treated on folic acid and hydroxyurea was admitted for new-onset diplopia day 0 D0 : refers to the start of the diplopia 6 weeks after respiratory tract infection due to rhinovirus. She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 .", "She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 . Brain MR angiography MRA showed a normal appearing circle of Willis. The cerebrospinal fluid CSF obtained by lumber puncture was normal WBC 1 cells/μl, RBC 0 cells/μl, glucose 2.9 mmol/L, protein 0.18 g/L, and absent oligoclonal bands . The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area.", "The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area. Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h.", "Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h. She was treated with five doses of IVMP 30 mg/kg/day followed by 9 days of oral prednisone 1 mg/kg/day . At discharge, her neurological exam was significant only for vertical diplopia. She presented 1 month later with 5 days of upper respiratory tract infection symptoms, fever, headache, and a rapidly progressive right-hand weakness D30 with normal alertness. She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir.", "She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir. White cell count was 13.4 × 10 9 /L, hemoglobin was 7.8 g/L, and platelets were 239 × 10 9 /L. While in the MRI machine D30 she deteriorated with vomiting and reduced level of consciousness Glasgow Coma Scale dropped from 15 to 8 over 30 min . Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F .", "Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F . The patient was immediately brought out of the magnet and her physical exam demonstrated unequal dilated pupils. She received IV mannitol and hypertonic saline for the management of acute intracranial hypertension/ herniation and was taken for surgery. She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis.", "She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis. Estimated blood loss was 3.5 L. She received 8 units of packed red blood cells, 3 units of cryoprecipitate, 6 units of fresh frozen plasma, and 3 units of platelets. Coagulation profile showed international normalization ratio = 3.38, prothrombin time = 51.2 s, and partial thromboplastin time = 122 s. An intraventricular pressure monitor was inserted. She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces.", "She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces. New white matter lesions were detected in the left posterior parietal and occipital lobes and in the left caudate head. MRI at D33 showed interval worsening with disseminated gray and white matter non-hemorrhagic lesions in the right cerebral and both cerebellar hemispheres, bilateral deep gray nuclei, as well as new necrotic non-hemorrhagic lesions in the left hemisphere Figures 2G-I . She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 .", "She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 . Prednisolone was tapered course over 6 weeks. At discharge D71 , she was able to say a few words and had better power of her right side. Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma.", "Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma. There was diffuse microglial activation and signs of early microinfarcts. Blood, CSF and urine culture, and PCR HSV1/2 were negative for bacteria and for viruses. CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative.", "CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative. Anti-extractable nuclear antigens SSA-RO, SSB-LA, smith, RNP were negative. Serum autoimmune antibodies panel NMO, NMDAR, AMPA I/II, GAB, MAG, VGCC, MOG, YO, HU, RI were negative but GAD antibody was slightly positive, possibly due to the IVIG infusion. EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange.", "EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange. Six months later, the patient was diagnosed to have Crohn's disease and primary sclerosing cholangitis. Two years later, the patient still suffers right hemiparesis but is able to walk without support. She presents an expressive aphasia. Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu .", "Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu . This mutation has not been previously reported in the HGMD database. This variant has been observed at a frequency of <0.01% across the entire Broad ExAC dataset of individuals without severe childhood onset disease 6/117,118 alleles . Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD .", "Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD . Several differential diagnoses of acute encephalopathy in a patient with sickle cell anemia can be considered. An infectious encephalitis, including herpes encephalitis, was ruled out by blood and CSF bacterial and viral cultures and negative HSV I/ II PCR. Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . .", "Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . . SCD patients are prone to ischemic or hemorrhagic strokes . . Primary hemorrhagic stroke is uncommon in pediatric SCD. Most cases were from adults and have been described in the context of previous ischemic stroke, aneurysms, low hemoglobin, acute chest syndrome, and hypertransfusions. Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . .", "Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . . This was ruled out as the MRI findings were not consistent with a specific vascular territory and normal arterial and venous flows were shown on vascular imaging. Another differential is posterior reversible encephalopathy syndrome which has been reported in SCD patients . . . . . However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury.", "However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury. However, it is associated to high ferritin and low triglycerides at the time of the encephalopathy, other multisystemic injuries, typical neuropathological findings, and recurrence over time, which were not noted in our patient . . Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia.", ". Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia. It caused punctate areas of hemorrhages in the basal ganglia, periventricular white matter, and mainly along the posterior parietal cortex. This was attributed to parvovirus B19-induced vasculitis . . In our patient, there was no sign of aplasia or any neuroradiological finding of parvovirus B19 infection. Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . .", "Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . . This was ruled out as the patient did not have clinical or radiological signs of acute chest syndrome or embolism and there was no arterial hypoxemia. Acute hemorrhagic encephalomyelitis has been described in pediatric patients following ADEM or ADEM-like episodes . . AHEM is the most plausible diagnosis in our patients based on the clinical and radiological presentation, the preceding ADEM-like episode, and the exclusion of other etiologies of acute encephalopathy. Other patients with AHEM have been described in the SCD context . .", "Other patients with AHEM have been described in the SCD context . . Many treatment options have been used to treat AHEM; of these, IV steroids have been associated with survival following aggressive, high-dose corticosteroid therapy . . . . . . . . . . Autosomal dominant mutations with incomplete penetrance in RANBP2 have been associated with susceptibility to infectioninduced necrotizing encephalopathy . . Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . .", "Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . . Our patient has a similar presentation and imaging features as infection-induced necrotizing encephalopathy, including bilateral thalamic involvement. The rare heterozygous previously unreported variant we identified in RANBP2 affects a very conserved aminoacid and is predicted deleterious using in silico tools a prediction tool performing a fast bioinformatics analysis which can predict the pathogenicity of a variant based on the change to an amino acid . It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy .", "It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy . making possible that both entities might be part of the same pathophysiological continuum. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells . . Hence, RANBP2 dysfunction might make neuronal cells much vulnerable to energy failure and necrosis when exposed to inflammatory or other stresses, such as those implicated in AHEM. This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication.", "This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication. All authors participated in gathering the data, designing the article, and discussing and editing the manuscript. aCKNoWleDgMeNts We thank Dr. S. Abish, Dr. N. Ahmed, and Mrs. C. Guiraut for their help. We are grateful to the Hoppenheim Fund from the Montreal Children Hospital Foundation. The first author of this article received a scholarship from the Hoppenheim Fund, Montreal Children Hospital Foundation .. This work was supported by grants from Heart and Stroke Foundation of Canada grant number: G-14-0005756 , and Foundation of Stars." ]	1,561	3,037	BACKGROUND: Acute hemorrhagic encephalomyelitis AHEM is considered as a rare form of acute disseminated encephalomyelitis characterized by fulminant encephalopathy with hemorrhagic necrosis and most often fatal outcome. OBJECTIVE: To report the association with Ran Binding Protein RANBP2 gene variant and the response to decompressive craniectomy and high-dose intravenous methylprednisolone IVMP in life-threatening AHEM. DESIGN: Single case study. CASE REPORT: A 6-year-old girl known to have sickle cell disease SCD presented an acquired demyelinating syndrome ADS with diplopia due to sudden unilateral fourth nerve palsy. She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma. Brain magnetic resonance imaging showed a left frontal necrotico-hemorrhagic lesion and new multifocal areas of demyelination. She underwent decompressive craniotomy and evacuation of an ongoing left frontoparietal hemorrhage. Comprehensive investigations ruled out vascular and infectious process. The neurological deterioration stopped concomitantly with combined neurosurgical drainage of the hematoma, decompressive craniotomy, IVMP, and intravenous immunoglobulins IVIG . She developed during the following months Crohn disease and sclerosing cholangitis. After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered. A heterozygous novel rare missense variant c.4993A>G, p.Lys1665Glu was identified in RANBP2, a gene associated with acute necrotizing encephalopathy. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP. This case report, in addition to others, stresses the likely efficacy of combined craniotomy, IVIG, and IVMP treatments in AHEM. RANBP2 mutations may sensitize the brain to inflammation and predispose to AHEM. Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system. It is usually fatal . . . . Many treatment options have been used including intravenous IV steroids, intravenous immunoglobulins IVIG , and plasmapheresis . . There have been few reports of survival following early intervention with high-dose corticosteroid therapy and/or decompressive craniotomy . . . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle. RANBP2 is associated with microtubules and mitochondria suggesting roles in intracellular protein trafficking or energy maintenance and homeostasis of neuronal cells. RANBP2 mutations have been reported in acute necrotizing encephalopathy ANE which could present with coma, convulsions, and encephalopathy. The hallmark of ANE is multiple, symmetric brain lesions located in the thalami bilaterally, putamina, deep periventricular white matter, cerebellum, and brainstem. It could be triggered by a viral infection in previously healthy children . . We report a new case of AHEM associated to a Ran Binding Protein RANBP -2 variant and responsive to combined craniectomy, intravenous methylprednisolone IVMP , and IVIG as inaugural manifestation of multisystemic autoimmunity in a girl with sickle cell disease SCD . A 6-year-old girl known for SCD treated on folic acid and hydroxyurea was admitted for new-onset diplopia day 0 D0 : refers to the start of the diplopia 6 weeks after respiratory tract infection due to rhinovirus. She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 . Brain MR angiography MRA showed a normal appearing circle of Willis. The cerebrospinal fluid CSF obtained by lumber puncture was normal WBC 1 cells/μl, RBC 0 cells/μl, glucose 2.9 mmol/L, protein 0.18 g/L, and absent oligoclonal bands . The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area. Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h. She was treated with five doses of IVMP 30 mg/kg/day followed by 9 days of oral prednisone 1 mg/kg/day . At discharge, her neurological exam was significant only for vertical diplopia. She presented 1 month later with 5 days of upper respiratory tract infection symptoms, fever, headache, and a rapidly progressive right-hand weakness D30 with normal alertness. She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir. White cell count was 13.4 × 10 9 /L, hemoglobin was 7.8 g/L, and platelets were 239 × 10 9 /L. While in the MRI machine D30 she deteriorated with vomiting and reduced level of consciousness Glasgow Coma Scale dropped from 15 to 8 over 30 min . Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F . The patient was immediately brought out of the magnet and her physical exam demonstrated unequal dilated pupils. She received IV mannitol and hypertonic saline for the management of acute intracranial hypertension/ herniation and was taken for surgery. She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis. Estimated blood loss was 3.5 L. She received 8 units of packed red blood cells, 3 units of cryoprecipitate, 6 units of fresh frozen plasma, and 3 units of platelets. Coagulation profile showed international normalization ratio = 3.38, prothrombin time = 51.2 s, and partial thromboplastin time = 122 s. An intraventricular pressure monitor was inserted. She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces. New white matter lesions were detected in the left posterior parietal and occipital lobes and in the left caudate head. MRI at D33 showed interval worsening with disseminated gray and white matter non-hemorrhagic lesions in the right cerebral and both cerebellar hemispheres, bilateral deep gray nuclei, as well as new necrotic non-hemorrhagic lesions in the left hemisphere Figures 2G-I . She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 . Prednisolone was tapered course over 6 weeks. At discharge D71 , she was able to say a few words and had better power of her right side. Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma. There was diffuse microglial activation and signs of early microinfarcts. Blood, CSF and urine culture, and PCR HSV1/2 were negative for bacteria and for viruses. CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative. Anti-extractable nuclear antigens SSA-RO, SSB-LA, smith, RNP were negative. Serum autoimmune antibodies panel NMO, NMDAR, AMPA I/II, GAB, MAG, VGCC, MOG, YO, HU, RI were negative but GAD antibody was slightly positive, possibly due to the IVIG infusion. EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange. Six months later, the patient was diagnosed to have Crohn's disease and primary sclerosing cholangitis. Two years later, the patient still suffers right hemiparesis but is able to walk without support. She presents an expressive aphasia. Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu . This mutation has not been previously reported in the HGMD database. This variant has been observed at a frequency of <0.01% across the entire Broad ExAC dataset of individuals without severe childhood onset disease 6/117,118 alleles . Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD . Several differential diagnoses of acute encephalopathy in a patient with sickle cell anemia can be considered. An infectious encephalitis, including herpes encephalitis, was ruled out by blood and CSF bacterial and viral cultures and negative HSV I/ II PCR. Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . . SCD patients are prone to ischemic or hemorrhagic strokes . . Primary hemorrhagic stroke is uncommon in pediatric SCD. Most cases were from adults and have been described in the context of previous ischemic stroke, aneurysms, low hemoglobin, acute chest syndrome, and hypertransfusions. Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . . This was ruled out as the MRI findings were not consistent with a specific vascular territory and normal arterial and venous flows were shown on vascular imaging. Another differential is posterior reversible encephalopathy syndrome which has been reported in SCD patients . . . . . However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury. However, it is associated to high ferritin and low triglycerides at the time of the encephalopathy, other multisystemic injuries, typical neuropathological findings, and recurrence over time, which were not noted in our patient . . Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia. It caused punctate areas of hemorrhages in the basal ganglia, periventricular white matter, and mainly along the posterior parietal cortex. This was attributed to parvovirus B19-induced vasculitis . . In our patient, there was no sign of aplasia or any neuroradiological finding of parvovirus B19 infection. Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . . This was ruled out as the patient did not have clinical or radiological signs of acute chest syndrome or embolism and there was no arterial hypoxemia. Acute hemorrhagic encephalomyelitis has been described in pediatric patients following ADEM or ADEM-like episodes . . AHEM is the most plausible diagnosis in our patients based on the clinical and radiological presentation, the preceding ADEM-like episode, and the exclusion of other etiologies of acute encephalopathy. Other patients with AHEM have been described in the SCD context . . Many treatment options have been used to treat AHEM; of these, IV steroids have been associated with survival following aggressive, high-dose corticosteroid therapy . . . . . . . . . . Autosomal dominant mutations with incomplete penetrance in RANBP2 have been associated with susceptibility to infectioninduced necrotizing encephalopathy . . Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . . Our patient has a similar presentation and imaging features as infection-induced necrotizing encephalopathy, including bilateral thalamic involvement. The rare heterozygous previously unreported variant we identified in RANBP2 affects a very conserved aminoacid and is predicted deleterious using in silico tools a prediction tool performing a fast bioinformatics analysis which can predict the pathogenicity of a variant based on the change to an amino acid . It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy . making possible that both entities might be part of the same pathophysiological continuum. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells . . Hence, RANBP2 dysfunction might make neuronal cells much vulnerable to energy failure and necrosis when exposed to inflammatory or other stresses, such as those implicated in AHEM. This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication. All authors participated in gathering the data, designing the article, and discussing and editing the manuscript. aCKNoWleDgMeNts We thank Dr. S. Abish, Dr. N. Ahmed, and Mrs. C. Guiraut for their help. We are grateful to the Hoppenheim Fund from the Montreal Children Hospital Foundation. The first author of this article received a scholarship from the Hoppenheim Fund, Montreal Children Hospital Foundation .. This work was supported by grants from Heart and Stroke Foundation of Canada grant number: G-14-0005756 , and Foundation of Stars.
When did she present with rapidly progressive right-hand weakness?	1 month later	[ "BACKGROUND: Acute hemorrhagic encephalomyelitis AHEM is considered as a rare form of acute disseminated encephalomyelitis characterized by fulminant encephalopathy with hemorrhagic necrosis and most often fatal outcome. OBJECTIVE: To report the association with Ran Binding Protein RANBP2 gene variant and the response to decompressive craniectomy and high-dose intravenous methylprednisolone IVMP in life-threatening AHEM. DESIGN: Single case study. CASE REPORT: A 6-year-old girl known to have sickle cell disease SCD presented an acquired demyelinating syndrome ADS with diplopia due to sudden unilateral fourth nerve palsy. She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma.", "She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma. Brain magnetic resonance imaging showed a left frontal necrotico-hemorrhagic lesion and new multifocal areas of demyelination. She underwent decompressive craniotomy and evacuation of an ongoing left frontoparietal hemorrhage. Comprehensive investigations ruled out vascular and infectious process. The neurological deterioration stopped concomitantly with combined neurosurgical drainage of the hematoma, decompressive craniotomy, IVMP, and intravenous immunoglobulins IVIG . She developed during the following months Crohn disease and sclerosing cholangitis. After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered.", "After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered. A heterozygous novel rare missense variant c.4993A>G, p.Lys1665Glu was identified in RANBP2, a gene associated with acute necrotizing encephalopathy. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP.", "RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP. This case report, in addition to others, stresses the likely efficacy of combined craniotomy, IVIG, and IVMP treatments in AHEM. RANBP2 mutations may sensitize the brain to inflammation and predispose to AHEM. Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system.", "Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system. It is usually fatal . . . . Many treatment options have been used including intravenous IV steroids, intravenous immunoglobulins IVIG , and plasmapheresis . . There have been few reports of survival following early intervention with high-dose corticosteroid therapy and/or decompressive craniotomy . . . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle.", ". . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle. RANBP2 is associated with microtubules and mitochondria suggesting roles in intracellular protein trafficking or energy maintenance and homeostasis of neuronal cells. RANBP2 mutations have been reported in acute necrotizing encephalopathy ANE which could present with coma, convulsions, and encephalopathy. The hallmark of ANE is multiple, symmetric brain lesions located in the thalami bilaterally, putamina, deep periventricular white matter, cerebellum, and brainstem. It could be triggered by a viral infection in previously healthy children . .", "It could be triggered by a viral infection in previously healthy children . . We report a new case of AHEM associated to a Ran Binding Protein RANBP -2 variant and responsive to combined craniectomy, intravenous methylprednisolone IVMP , and IVIG as inaugural manifestation of multisystemic autoimmunity in a girl with sickle cell disease SCD . A 6-year-old girl known for SCD treated on folic acid and hydroxyurea was admitted for new-onset diplopia day 0 D0 : refers to the start of the diplopia 6 weeks after respiratory tract infection due to rhinovirus. She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 .", "She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 . Brain MR angiography MRA showed a normal appearing circle of Willis. The cerebrospinal fluid CSF obtained by lumber puncture was normal WBC 1 cells/μl, RBC 0 cells/μl, glucose 2.9 mmol/L, protein 0.18 g/L, and absent oligoclonal bands . The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area.", "The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area. Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h.", "Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h. She was treated with five doses of IVMP 30 mg/kg/day followed by 9 days of oral prednisone 1 mg/kg/day . At discharge, her neurological exam was significant only for vertical diplopia. She presented 1 month later with 5 days of upper respiratory tract infection symptoms, fever, headache, and a rapidly progressive right-hand weakness D30 with normal alertness. She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir.", "She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir. White cell count was 13.4 × 10 9 /L, hemoglobin was 7.8 g/L, and platelets were 239 × 10 9 /L. While in the MRI machine D30 she deteriorated with vomiting and reduced level of consciousness Glasgow Coma Scale dropped from 15 to 8 over 30 min . Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F .", "Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F . The patient was immediately brought out of the magnet and her physical exam demonstrated unequal dilated pupils. She received IV mannitol and hypertonic saline for the management of acute intracranial hypertension/ herniation and was taken for surgery. She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis.", "She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis. Estimated blood loss was 3.5 L. She received 8 units of packed red blood cells, 3 units of cryoprecipitate, 6 units of fresh frozen plasma, and 3 units of platelets. Coagulation profile showed international normalization ratio = 3.38, prothrombin time = 51.2 s, and partial thromboplastin time = 122 s. An intraventricular pressure monitor was inserted. She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces.", "She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces. New white matter lesions were detected in the left posterior parietal and occipital lobes and in the left caudate head. MRI at D33 showed interval worsening with disseminated gray and white matter non-hemorrhagic lesions in the right cerebral and both cerebellar hemispheres, bilateral deep gray nuclei, as well as new necrotic non-hemorrhagic lesions in the left hemisphere Figures 2G-I . She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 .", "She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 . Prednisolone was tapered course over 6 weeks. At discharge D71 , she was able to say a few words and had better power of her right side. Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma.", "Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma. There was diffuse microglial activation and signs of early microinfarcts. Blood, CSF and urine culture, and PCR HSV1/2 were negative for bacteria and for viruses. CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative.", "CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative. Anti-extractable nuclear antigens SSA-RO, SSB-LA, smith, RNP were negative. Serum autoimmune antibodies panel NMO, NMDAR, AMPA I/II, GAB, MAG, VGCC, MOG, YO, HU, RI were negative but GAD antibody was slightly positive, possibly due to the IVIG infusion. EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange.", "EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange. Six months later, the patient was diagnosed to have Crohn's disease and primary sclerosing cholangitis. Two years later, the patient still suffers right hemiparesis but is able to walk without support. She presents an expressive aphasia. Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu .", "Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu . This mutation has not been previously reported in the HGMD database. This variant has been observed at a frequency of <0.01% across the entire Broad ExAC dataset of individuals without severe childhood onset disease 6/117,118 alleles . Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD .", "Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD . Several differential diagnoses of acute encephalopathy in a patient with sickle cell anemia can be considered. An infectious encephalitis, including herpes encephalitis, was ruled out by blood and CSF bacterial and viral cultures and negative HSV I/ II PCR. Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . .", "Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . . SCD patients are prone to ischemic or hemorrhagic strokes . . Primary hemorrhagic stroke is uncommon in pediatric SCD. Most cases were from adults and have been described in the context of previous ischemic stroke, aneurysms, low hemoglobin, acute chest syndrome, and hypertransfusions. Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . .", "Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . . This was ruled out as the MRI findings were not consistent with a specific vascular territory and normal arterial and venous flows were shown on vascular imaging. Another differential is posterior reversible encephalopathy syndrome which has been reported in SCD patients . . . . . However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury.", "However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury. However, it is associated to high ferritin and low triglycerides at the time of the encephalopathy, other multisystemic injuries, typical neuropathological findings, and recurrence over time, which were not noted in our patient . . Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia.", ". Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia. It caused punctate areas of hemorrhages in the basal ganglia, periventricular white matter, and mainly along the posterior parietal cortex. This was attributed to parvovirus B19-induced vasculitis . . In our patient, there was no sign of aplasia or any neuroradiological finding of parvovirus B19 infection. Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . .", "Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . . This was ruled out as the patient did not have clinical or radiological signs of acute chest syndrome or embolism and there was no arterial hypoxemia. Acute hemorrhagic encephalomyelitis has been described in pediatric patients following ADEM or ADEM-like episodes . . AHEM is the most plausible diagnosis in our patients based on the clinical and radiological presentation, the preceding ADEM-like episode, and the exclusion of other etiologies of acute encephalopathy. Other patients with AHEM have been described in the SCD context . .", "Other patients with AHEM have been described in the SCD context . . Many treatment options have been used to treat AHEM; of these, IV steroids have been associated with survival following aggressive, high-dose corticosteroid therapy . . . . . . . . . . Autosomal dominant mutations with incomplete penetrance in RANBP2 have been associated with susceptibility to infectioninduced necrotizing encephalopathy . . Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . .", "Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . . Our patient has a similar presentation and imaging features as infection-induced necrotizing encephalopathy, including bilateral thalamic involvement. The rare heterozygous previously unreported variant we identified in RANBP2 affects a very conserved aminoacid and is predicted deleterious using in silico tools a prediction tool performing a fast bioinformatics analysis which can predict the pathogenicity of a variant based on the change to an amino acid . It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy .", "It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy . making possible that both entities might be part of the same pathophysiological continuum. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells . . Hence, RANBP2 dysfunction might make neuronal cells much vulnerable to energy failure and necrosis when exposed to inflammatory or other stresses, such as those implicated in AHEM. This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication.", "This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication. All authors participated in gathering the data, designing the article, and discussing and editing the manuscript. aCKNoWleDgMeNts We thank Dr. S. Abish, Dr. N. Ahmed, and Mrs. C. Guiraut for their help. We are grateful to the Hoppenheim Fund from the Montreal Children Hospital Foundation. The first author of this article received a scholarship from the Hoppenheim Fund, Montreal Children Hospital Foundation .. This work was supported by grants from Heart and Stroke Foundation of Canada grant number: G-14-0005756 , and Foundation of Stars." ]	1,561	3,038	BACKGROUND: Acute hemorrhagic encephalomyelitis AHEM is considered as a rare form of acute disseminated encephalomyelitis characterized by fulminant encephalopathy with hemorrhagic necrosis and most often fatal outcome. OBJECTIVE: To report the association with Ran Binding Protein RANBP2 gene variant and the response to decompressive craniectomy and high-dose intravenous methylprednisolone IVMP in life-threatening AHEM. DESIGN: Single case study. CASE REPORT: A 6-year-old girl known to have sickle cell disease SCD presented an acquired demyelinating syndrome ADS with diplopia due to sudden unilateral fourth nerve palsy. She received five pulses of IVMP 30 mg/kg/day . Two weeks after steroid weaning, she developed right hemiplegia and coma. Brain magnetic resonance imaging showed a left frontal necrotico-hemorrhagic lesion and new multifocal areas of demyelination. She underwent decompressive craniotomy and evacuation of an ongoing left frontoparietal hemorrhage. Comprehensive investigations ruled out vascular and infectious process. The neurological deterioration stopped concomitantly with combined neurosurgical drainage of the hematoma, decompressive craniotomy, IVMP, and intravenous immunoglobulins IVIG . She developed during the following months Crohn disease and sclerosing cholangitis. After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered. A heterozygous novel rare missense variant c.4993A>G, p.Lys1665Glu was identified in RANBP2, a gene associated with acute necrotizing encephalopathy. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP. This case report, in addition to others, stresses the likely efficacy of combined craniotomy, IVIG, and IVMP treatments in AHEM. RANBP2 mutations may sensitize the brain to inflammation and predispose to AHEM. Text: Acute hemorrhagic encephalomyelitis AHEM or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis ADEM . AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system. It is usually fatal . . . . Many treatment options have been used including intravenous IV steroids, intravenous immunoglobulins IVIG , and plasmapheresis . . There have been few reports of survival following early intervention with high-dose corticosteroid therapy and/or decompressive craniotomy . . . . . . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle. RANBP2 is associated with microtubules and mitochondria suggesting roles in intracellular protein trafficking or energy maintenance and homeostasis of neuronal cells. RANBP2 mutations have been reported in acute necrotizing encephalopathy ANE which could present with coma, convulsions, and encephalopathy. The hallmark of ANE is multiple, symmetric brain lesions located in the thalami bilaterally, putamina, deep periventricular white matter, cerebellum, and brainstem. It could be triggered by a viral infection in previously healthy children . . We report a new case of AHEM associated to a Ran Binding Protein RANBP -2 variant and responsive to combined craniectomy, intravenous methylprednisolone IVMP , and IVIG as inaugural manifestation of multisystemic autoimmunity in a girl with sickle cell disease SCD . A 6-year-old girl known for SCD treated on folic acid and hydroxyurea was admitted for new-onset diplopia day 0 D0 : refers to the start of the diplopia 6 weeks after respiratory tract infection due to rhinovirus. She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging MRI performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal Figure 1 . Brain MR angiography MRA showed a normal appearing circle of Willis. The cerebrospinal fluid CSF obtained by lumber puncture was normal WBC 1 cells/μl, RBC 0 cells/μl, glucose 2.9 mmol/L, protein 0.18 g/L, and absent oligoclonal bands . The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus , and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive 1:1,280 reflecting a previously acquired common and self-limited infection in our area. Antinuclear antibodies ANA were positive 1:160 . B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h. She was treated with five doses of IVMP 30 mg/kg/day followed by 9 days of oral prednisone 1 mg/kg/day . At discharge, her neurological exam was significant only for vertical diplopia. She presented 1 month later with 5 days of upper respiratory tract infection symptoms, fever, headache, and a rapidly progressive right-hand weakness D30 with normal alertness. She had normal blood pressure 120/81 mmHg . She was started on cefotaxime, vancomycin, and acyclovir. White cell count was 13.4 × 10 9 /L, hemoglobin was 7.8 g/L, and platelets were 239 × 10 9 /L. While in the MRI machine D30 she deteriorated with vomiting and reduced level of consciousness Glasgow Coma Scale dropped from 15 to 8 over 30 min . Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal Figures 2A-F . The patient was immediately brought out of the magnet and her physical exam demonstrated unequal dilated pupils. She received IV mannitol and hypertonic saline for the management of acute intracranial hypertension/ herniation and was taken for surgery. She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain EVD . Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis. Estimated blood loss was 3.5 L. She received 8 units of packed red blood cells, 3 units of cryoprecipitate, 6 units of fresh frozen plasma, and 3 units of platelets. Coagulation profile showed international normalization ratio = 3.38, prothrombin time = 51.2 s, and partial thromboplastin time = 122 s. An intraventricular pressure monitor was inserted. She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces. New white matter lesions were detected in the left posterior parietal and occipital lobes and in the left caudate head. MRI at D33 showed interval worsening with disseminated gray and white matter non-hemorrhagic lesions in the right cerebral and both cerebellar hemispheres, bilateral deep gray nuclei, as well as new necrotic non-hemorrhagic lesions in the left hemisphere Figures 2G-I . She was started on IVMP 30 mg/kg/ day for 5 days and IVIG 1 g/kg/day for 2 days . Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions Figure 3 . Prednisolone was tapered course over 6 weeks. At discharge D71 , she was able to say a few words and had better power of her right side. Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma. There was diffuse microglial activation and signs of early microinfarcts. Blood, CSF and urine culture, and PCR HSV1/2 were negative for bacteria and for viruses. CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative. Anti-extractable nuclear antigens SSA-RO, SSB-LA, smith, RNP were negative. Serum autoimmune antibodies panel NMO, NMDAR, AMPA I/II, GAB, MAG, VGCC, MOG, YO, HU, RI were negative but GAD antibody was slightly positive, possibly due to the IVIG infusion. EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange. Six months later, the patient was diagnosed to have Crohn's disease and primary sclerosing cholangitis. Two years later, the patient still suffers right hemiparesis but is able to walk without support. She presents an expressive aphasia. Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel Table 1 targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation NM_006267.4, c.4993A>G, p.Lys1665Glu . This mutation has not been previously reported in the HGMD database. This variant has been observed at a frequency of <0.01% across the entire Broad ExAC dataset of individuals without severe childhood onset disease 6/117,118 alleles . Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging SIFT and Align GVGD . Several differential diagnoses of acute encephalopathy in a patient with sickle cell anemia can be considered. An infectious encephalitis, including herpes encephalitis, was ruled out by blood and CSF bacterial and viral cultures and negative HSV I/ II PCR. Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A . . SCD patients are prone to ischemic or hemorrhagic strokes . . Primary hemorrhagic stroke is uncommon in pediatric SCD. Most cases were from adults and have been described in the context of previous ischemic stroke, aneurysms, low hemoglobin, acute chest syndrome, and hypertransfusions. Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case . . This was ruled out as the MRI findings were not consistent with a specific vascular territory and normal arterial and venous flows were shown on vascular imaging. Another differential is posterior reversible encephalopathy syndrome which has been reported in SCD patients . . . . . However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury. However, it is associated to high ferritin and low triglycerides at the time of the encephalopathy, other multisystemic injuries, typical neuropathological findings, and recurrence over time, which were not noted in our patient . . Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia. It caused punctate areas of hemorrhages in the basal ganglia, periventricular white matter, and mainly along the posterior parietal cortex. This was attributed to parvovirus B19-induced vasculitis . . In our patient, there was no sign of aplasia or any neuroradiological finding of parvovirus B19 infection. Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia . . This was ruled out as the patient did not have clinical or radiological signs of acute chest syndrome or embolism and there was no arterial hypoxemia. Acute hemorrhagic encephalomyelitis has been described in pediatric patients following ADEM or ADEM-like episodes . . AHEM is the most plausible diagnosis in our patients based on the clinical and radiological presentation, the preceding ADEM-like episode, and the exclusion of other etiologies of acute encephalopathy. Other patients with AHEM have been described in the SCD context . . Many treatment options have been used to treat AHEM; of these, IV steroids have been associated with survival following aggressive, high-dose corticosteroid therapy . . . . . . . . . . Autosomal dominant mutations with incomplete penetrance in RANBP2 have been associated with susceptibility to infectioninduced necrotizing encephalopathy . . Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum . . Our patient has a similar presentation and imaging features as infection-induced necrotizing encephalopathy, including bilateral thalamic involvement. The rare heterozygous previously unreported variant we identified in RANBP2 affects a very conserved aminoacid and is predicted deleterious using in silico tools a prediction tool performing a fast bioinformatics analysis which can predict the pathogenicity of a variant based on the change to an amino acid . It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy . making possible that both entities might be part of the same pathophysiological continuum. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells . . Hence, RANBP2 dysfunction might make neuronal cells much vulnerable to energy failure and necrosis when exposed to inflammatory or other stresses, such as those implicated in AHEM. This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication. All authors participated in gathering the data, designing the article, and discussing and editing the manuscript. aCKNoWleDgMeNts We thank Dr. S. Abish, Dr. N. Ahmed, and Mrs. C. Guiraut for their help. We are grateful to the Hoppenheim Fund from the Montreal Children Hospital Foundation. The first author of this article received a scholarship from the Hoppenheim Fund, Montreal Children Hospital Foundation .. This work was supported by grants from Heart and Stroke Foundation of Canada grant number: G-14-0005756 , and Foundation of Stars.
How many samples were obtained?	11,399	[ "BACKGROUND: Human bocavirus 1 HBoV1 is an important cause of acute respiratory illness ARI , yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients ≤14 years old , with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection.", "RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients 45.2%, 112/248 . A significant difference in the prevalence of HBoV1 was found in patients in different age groups p < 0.001 , and the peak prevalence was found in patients aged 7–12 months 4.7%, 56/1203 . Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature.", "Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide.", "Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness ARI , according to different studies . Serological and nucleic acid test results are generally consistent , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms .", "HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications . In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases.", "In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus.", "The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children ≤14 years old hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ .", "This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ . Inclusion criteria were pediatric patients ≤14 years old who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously .", "Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature °C , mean relative humidity % , rainfall mm , mean wind speed m/s , mean air pressure hPa , mean vapor pressure hPa , sunshine duration h . Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit Qiagen, Shanghai, China , respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported .", "Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 SPSS Inc., Chicago, IL, USA . Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016.", "All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 7361:4038 and the median age was 1.75 years interquartile range 0.75-3.83 . Overall, 86.5% 9857/11399 of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 .", "All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 . Two hundred forty-eight of 11,399 patients 2.2% tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 45.2% were co-infected with other pathogens, most frequently with RSV 11.7%, 29/248 Table 1 . The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 .", "The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 . To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups p < 0.001 and the peak prevalence was found in patients aged 7-12 months 4.7%, 56/1203 Fig. 1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1.", "1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′ , has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C mean ± standard deviation , humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 .", "Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure adjusted R 2 = 0.793, p < 0.001 and mean vapor pressure adjusted R 2 = 0.929, p < 0.001 , which linearly associated with mean temperature, and rainfall adjusted R 2 = 0.278, p < 0.001 , which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively.", "The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature coefficient = 0.259 in the current temperature model p = 0.002 , coefficient = 0.328 in mean temperature in the preceding month model p < 0.001 . Conversely, HBoV1 prevalence was negatively correlated with relative humidity coefficient = − 0.126 in the current temperature model p = 0.024 , coefficient = − 0.083 in the temperature delay model p = 0.039 Table 2 . ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 .", "ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports .", "To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports . Overall, 49.2% 5606/11399 of patients tested positive for one or more respiratory pathogens, 2.2% 248/11399 of patients were tested with HBoV1 infection Table 1 . A higher prevalence of HBoV1 was detected in male patients compared with female patients p = 0.019 , consistent with previous reports . Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 .", "Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 . This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference Fig. 2 . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this.", "Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages p < 0.001 . The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 .", "The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region.", "Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period Fig. 3 . In this study, two HBoV1 peaks were observed Fig. 2 . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 .", "The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring , as well as from tropical regions, such as India, where no obvious epidemic season has been found . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model.", "To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity Table 2 . In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 .", "Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 .", "It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study p > 0.05 Table 2 . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation.", "First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections." ]	1,573	3,267	BACKGROUND: Human bocavirus 1 HBoV1 is an important cause of acute respiratory illness ARI , yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients ≤14 years old , with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients 45.2%, 112/248 . A significant difference in the prevalence of HBoV1 was found in patients in different age groups p < 0.001 , and the peak prevalence was found in patients aged 7–12 months 4.7%, 56/1203 . Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness ARI , according to different studies . Serological and nucleic acid test results are generally consistent , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications . In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children ≤14 years old hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ . Inclusion criteria were pediatric patients ≤14 years old who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature °C , mean relative humidity % , rainfall mm , mean wind speed m/s , mean air pressure hPa , mean vapor pressure hPa , sunshine duration h . Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit Qiagen, Shanghai, China , respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 SPSS Inc., Chicago, IL, USA . Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 7361:4038 and the median age was 1.75 years interquartile range 0.75-3.83 . Overall, 86.5% 9857/11399 of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 . Two hundred forty-eight of 11,399 patients 2.2% tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 45.2% were co-infected with other pathogens, most frequently with RSV 11.7%, 29/248 Table 1 . The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 . To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups p < 0.001 and the peak prevalence was found in patients aged 7-12 months 4.7%, 56/1203 Fig. 1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′ , has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C mean ± standard deviation , humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure adjusted R 2 = 0.793, p < 0.001 and mean vapor pressure adjusted R 2 = 0.929, p < 0.001 , which linearly associated with mean temperature, and rainfall adjusted R 2 = 0.278, p < 0.001 , which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature coefficient = 0.259 in the current temperature model p = 0.002 , coefficient = 0.328 in mean temperature in the preceding month model p < 0.001 . Conversely, HBoV1 prevalence was negatively correlated with relative humidity coefficient = − 0.126 in the current temperature model p = 0.024 , coefficient = − 0.083 in the temperature delay model p = 0.039 Table 2 . ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports . Overall, 49.2% 5606/11399 of patients tested positive for one or more respiratory pathogens, 2.2% 248/11399 of patients were tested with HBoV1 infection Table 1 . A higher prevalence of HBoV1 was detected in male patients compared with female patients p = 0.019 , consistent with previous reports . Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 . This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference Fig. 2 . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages p < 0.001 . The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period Fig. 3 . In this study, two HBoV1 peaks were observed Fig. 2 . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring , as well as from tropical regions, such as India, where no obvious epidemic season has been found . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity Table 2 . In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study p > 0.05 Table 2 . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections.
What percentage of patients were positive for at least one respiratory pathogen?	49.2%	[ "BACKGROUND: Human bocavirus 1 HBoV1 is an important cause of acute respiratory illness ARI , yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients ≤14 years old , with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection.", "RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients 45.2%, 112/248 . A significant difference in the prevalence of HBoV1 was found in patients in different age groups p < 0.001 , and the peak prevalence was found in patients aged 7–12 months 4.7%, 56/1203 . Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature.", "Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide.", "Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness ARI , according to different studies . Serological and nucleic acid test results are generally consistent , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms .", "HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications . In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases.", "In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus.", "The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children ≤14 years old hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ .", "This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ . Inclusion criteria were pediatric patients ≤14 years old who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously .", "Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature °C , mean relative humidity % , rainfall mm , mean wind speed m/s , mean air pressure hPa , mean vapor pressure hPa , sunshine duration h . Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit Qiagen, Shanghai, China , respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported .", "Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 SPSS Inc., Chicago, IL, USA . Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016.", "All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 7361:4038 and the median age was 1.75 years interquartile range 0.75-3.83 . Overall, 86.5% 9857/11399 of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 .", "All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 . Two hundred forty-eight of 11,399 patients 2.2% tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 45.2% were co-infected with other pathogens, most frequently with RSV 11.7%, 29/248 Table 1 . The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 .", "The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 . To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups p < 0.001 and the peak prevalence was found in patients aged 7-12 months 4.7%, 56/1203 Fig. 1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1.", "1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′ , has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C mean ± standard deviation , humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 .", "Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure adjusted R 2 = 0.793, p < 0.001 and mean vapor pressure adjusted R 2 = 0.929, p < 0.001 , which linearly associated with mean temperature, and rainfall adjusted R 2 = 0.278, p < 0.001 , which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively.", "The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature coefficient = 0.259 in the current temperature model p = 0.002 , coefficient = 0.328 in mean temperature in the preceding month model p < 0.001 . Conversely, HBoV1 prevalence was negatively correlated with relative humidity coefficient = − 0.126 in the current temperature model p = 0.024 , coefficient = − 0.083 in the temperature delay model p = 0.039 Table 2 . ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 .", "ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports .", "To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports . Overall, 49.2% 5606/11399 of patients tested positive for one or more respiratory pathogens, 2.2% 248/11399 of patients were tested with HBoV1 infection Table 1 . A higher prevalence of HBoV1 was detected in male patients compared with female patients p = 0.019 , consistent with previous reports . Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 .", "Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 . This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference Fig. 2 . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this.", "Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages p < 0.001 . The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 .", "The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region.", "Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period Fig. 3 . In this study, two HBoV1 peaks were observed Fig. 2 . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 .", "The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring , as well as from tropical regions, such as India, where no obvious epidemic season has been found . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model.", "To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity Table 2 . In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 .", "Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 .", "It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study p > 0.05 Table 2 . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation.", "First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections." ]	1,573	3,268	BACKGROUND: Human bocavirus 1 HBoV1 is an important cause of acute respiratory illness ARI , yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients ≤14 years old , with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients 45.2%, 112/248 . A significant difference in the prevalence of HBoV1 was found in patients in different age groups p < 0.001 , and the peak prevalence was found in patients aged 7–12 months 4.7%, 56/1203 . Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness ARI , according to different studies . Serological and nucleic acid test results are generally consistent , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications . In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children ≤14 years old hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ . Inclusion criteria were pediatric patients ≤14 years old who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature °C , mean relative humidity % , rainfall mm , mean wind speed m/s , mean air pressure hPa , mean vapor pressure hPa , sunshine duration h . Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit Qiagen, Shanghai, China , respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 SPSS Inc., Chicago, IL, USA . Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 7361:4038 and the median age was 1.75 years interquartile range 0.75-3.83 . Overall, 86.5% 9857/11399 of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 . Two hundred forty-eight of 11,399 patients 2.2% tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 45.2% were co-infected with other pathogens, most frequently with RSV 11.7%, 29/248 Table 1 . The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 . To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups p < 0.001 and the peak prevalence was found in patients aged 7-12 months 4.7%, 56/1203 Fig. 1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′ , has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C mean ± standard deviation , humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure adjusted R 2 = 0.793, p < 0.001 and mean vapor pressure adjusted R 2 = 0.929, p < 0.001 , which linearly associated with mean temperature, and rainfall adjusted R 2 = 0.278, p < 0.001 , which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature coefficient = 0.259 in the current temperature model p = 0.002 , coefficient = 0.328 in mean temperature in the preceding month model p < 0.001 . Conversely, HBoV1 prevalence was negatively correlated with relative humidity coefficient = − 0.126 in the current temperature model p = 0.024 , coefficient = − 0.083 in the temperature delay model p = 0.039 Table 2 . ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports . Overall, 49.2% 5606/11399 of patients tested positive for one or more respiratory pathogens, 2.2% 248/11399 of patients were tested with HBoV1 infection Table 1 . A higher prevalence of HBoV1 was detected in male patients compared with female patients p = 0.019 , consistent with previous reports . Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 . This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference Fig. 2 . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages p < 0.001 . The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period Fig. 3 . In this study, two HBoV1 peaks were observed Fig. 2 . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring , as well as from tropical regions, such as India, where no obvious epidemic season has been found . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity Table 2 . In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study p > 0.05 Table 2 . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections.
What percentage of patients tested positive for HBoV1?	2.2%	[ "BACKGROUND: Human bocavirus 1 HBoV1 is an important cause of acute respiratory illness ARI , yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients ≤14 years old , with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection.", "RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients 45.2%, 112/248 . A significant difference in the prevalence of HBoV1 was found in patients in different age groups p < 0.001 , and the peak prevalence was found in patients aged 7–12 months 4.7%, 56/1203 . Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature.", "Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide.", "Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness ARI , according to different studies . Serological and nucleic acid test results are generally consistent , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms .", "HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications . In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases.", "In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus.", "The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children ≤14 years old hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ .", "This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ . Inclusion criteria were pediatric patients ≤14 years old who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously .", "Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature °C , mean relative humidity % , rainfall mm , mean wind speed m/s , mean air pressure hPa , mean vapor pressure hPa , sunshine duration h . Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit Qiagen, Shanghai, China , respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported .", "Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 SPSS Inc., Chicago, IL, USA . Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016.", "All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 7361:4038 and the median age was 1.75 years interquartile range 0.75-3.83 . Overall, 86.5% 9857/11399 of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 .", "All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 . Two hundred forty-eight of 11,399 patients 2.2% tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 45.2% were co-infected with other pathogens, most frequently with RSV 11.7%, 29/248 Table 1 . The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 .", "The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 . To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups p < 0.001 and the peak prevalence was found in patients aged 7-12 months 4.7%, 56/1203 Fig. 1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1.", "1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′ , has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C mean ± standard deviation , humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 .", "Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure adjusted R 2 = 0.793, p < 0.001 and mean vapor pressure adjusted R 2 = 0.929, p < 0.001 , which linearly associated with mean temperature, and rainfall adjusted R 2 = 0.278, p < 0.001 , which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively.", "The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature coefficient = 0.259 in the current temperature model p = 0.002 , coefficient = 0.328 in mean temperature in the preceding month model p < 0.001 . Conversely, HBoV1 prevalence was negatively correlated with relative humidity coefficient = − 0.126 in the current temperature model p = 0.024 , coefficient = − 0.083 in the temperature delay model p = 0.039 Table 2 . ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 .", "ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports .", "To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports . Overall, 49.2% 5606/11399 of patients tested positive for one or more respiratory pathogens, 2.2% 248/11399 of patients were tested with HBoV1 infection Table 1 . A higher prevalence of HBoV1 was detected in male patients compared with female patients p = 0.019 , consistent with previous reports . Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 .", "Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 . This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference Fig. 2 . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this.", "Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages p < 0.001 . The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 .", "The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region.", "Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period Fig. 3 . In this study, two HBoV1 peaks were observed Fig. 2 . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 .", "The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring , as well as from tropical regions, such as India, where no obvious epidemic season has been found . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model.", "To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity Table 2 . In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 .", "Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 .", "It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study p > 0.05 Table 2 . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation.", "First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections." ]	1,573	3,269	BACKGROUND: Human bocavirus 1 HBoV1 is an important cause of acute respiratory illness ARI , yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients ≤14 years old , with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients 45.2%, 112/248 . A significant difference in the prevalence of HBoV1 was found in patients in different age groups p < 0.001 , and the peak prevalence was found in patients aged 7–12 months 4.7%, 56/1203 . Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness ARI , according to different studies . Serological and nucleic acid test results are generally consistent , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications . In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children ≤14 years old hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ . Inclusion criteria were pediatric patients ≤14 years old who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature °C , mean relative humidity % , rainfall mm , mean wind speed m/s , mean air pressure hPa , mean vapor pressure hPa , sunshine duration h . Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit Qiagen, Shanghai, China , respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 SPSS Inc., Chicago, IL, USA . Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 7361:4038 and the median age was 1.75 years interquartile range 0.75-3.83 . Overall, 86.5% 9857/11399 of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 . Two hundred forty-eight of 11,399 patients 2.2% tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 45.2% were co-infected with other pathogens, most frequently with RSV 11.7%, 29/248 Table 1 . The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 . To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups p < 0.001 and the peak prevalence was found in patients aged 7-12 months 4.7%, 56/1203 Fig. 1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′ , has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C mean ± standard deviation , humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure adjusted R 2 = 0.793, p < 0.001 and mean vapor pressure adjusted R 2 = 0.929, p < 0.001 , which linearly associated with mean temperature, and rainfall adjusted R 2 = 0.278, p < 0.001 , which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature coefficient = 0.259 in the current temperature model p = 0.002 , coefficient = 0.328 in mean temperature in the preceding month model p < 0.001 . Conversely, HBoV1 prevalence was negatively correlated with relative humidity coefficient = − 0.126 in the current temperature model p = 0.024 , coefficient = − 0.083 in the temperature delay model p = 0.039 Table 2 . ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports . Overall, 49.2% 5606/11399 of patients tested positive for one or more respiratory pathogens, 2.2% 248/11399 of patients were tested with HBoV1 infection Table 1 . A higher prevalence of HBoV1 was detected in male patients compared with female patients p = 0.019 , consistent with previous reports . Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 . This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference Fig. 2 . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages p < 0.001 . The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period Fig. 3 . In this study, two HBoV1 peaks were observed Fig. 2 . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring , as well as from tropical regions, such as India, where no obvious epidemic season has been found . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity Table 2 . In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study p > 0.05 Table 2 . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections.
When was HBoV1 first identified?	2005	[ "BACKGROUND: Human bocavirus 1 HBoV1 is an important cause of acute respiratory illness ARI , yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients ≤14 years old , with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection.", "RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients 45.2%, 112/248 . A significant difference in the prevalence of HBoV1 was found in patients in different age groups p < 0.001 , and the peak prevalence was found in patients aged 7–12 months 4.7%, 56/1203 . Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature.", "Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide.", "Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness ARI , according to different studies . Serological and nucleic acid test results are generally consistent , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms .", "HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications . In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases.", "In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus.", "The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children ≤14 years old hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ .", "This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ . Inclusion criteria were pediatric patients ≤14 years old who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously .", "Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature °C , mean relative humidity % , rainfall mm , mean wind speed m/s , mean air pressure hPa , mean vapor pressure hPa , sunshine duration h . Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit Qiagen, Shanghai, China , respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported .", "Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 SPSS Inc., Chicago, IL, USA . Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016.", "All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 7361:4038 and the median age was 1.75 years interquartile range 0.75-3.83 . Overall, 86.5% 9857/11399 of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 .", "All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 . Two hundred forty-eight of 11,399 patients 2.2% tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 45.2% were co-infected with other pathogens, most frequently with RSV 11.7%, 29/248 Table 1 . The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 .", "The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 . To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups p < 0.001 and the peak prevalence was found in patients aged 7-12 months 4.7%, 56/1203 Fig. 1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1.", "1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′ , has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C mean ± standard deviation , humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 .", "Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure adjusted R 2 = 0.793, p < 0.001 and mean vapor pressure adjusted R 2 = 0.929, p < 0.001 , which linearly associated with mean temperature, and rainfall adjusted R 2 = 0.278, p < 0.001 , which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively.", "The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature coefficient = 0.259 in the current temperature model p = 0.002 , coefficient = 0.328 in mean temperature in the preceding month model p < 0.001 . Conversely, HBoV1 prevalence was negatively correlated with relative humidity coefficient = − 0.126 in the current temperature model p = 0.024 , coefficient = − 0.083 in the temperature delay model p = 0.039 Table 2 . ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 .", "ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports .", "To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports . Overall, 49.2% 5606/11399 of patients tested positive for one or more respiratory pathogens, 2.2% 248/11399 of patients were tested with HBoV1 infection Table 1 . A higher prevalence of HBoV1 was detected in male patients compared with female patients p = 0.019 , consistent with previous reports . Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 .", "Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 . This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference Fig. 2 . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this.", "Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages p < 0.001 . The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 .", "The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region.", "Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period Fig. 3 . In this study, two HBoV1 peaks were observed Fig. 2 . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 .", "The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring , as well as from tropical regions, such as India, where no obvious epidemic season has been found . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model.", "To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity Table 2 . In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 .", "Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 .", "It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study p > 0.05 Table 2 . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation.", "First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections." ]	1,573	3,270	BACKGROUND: Human bocavirus 1 HBoV1 is an important cause of acute respiratory illness ARI , yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients ≤14 years old , with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients 45.2%, 112/248 . A significant difference in the prevalence of HBoV1 was found in patients in different age groups p < 0.001 , and the peak prevalence was found in patients aged 7–12 months 4.7%, 56/1203 . Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness ARI , according to different studies . Serological and nucleic acid test results are generally consistent , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications . In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children ≤14 years old hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ . Inclusion criteria were pediatric patients ≤14 years old who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature °C , mean relative humidity % , rainfall mm , mean wind speed m/s , mean air pressure hPa , mean vapor pressure hPa , sunshine duration h . Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit Qiagen, Shanghai, China , respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 SPSS Inc., Chicago, IL, USA . Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 7361:4038 and the median age was 1.75 years interquartile range 0.75-3.83 . Overall, 86.5% 9857/11399 of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 . Two hundred forty-eight of 11,399 patients 2.2% tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 45.2% were co-infected with other pathogens, most frequently with RSV 11.7%, 29/248 Table 1 . The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 . To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups p < 0.001 and the peak prevalence was found in patients aged 7-12 months 4.7%, 56/1203 Fig. 1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′ , has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C mean ± standard deviation , humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure adjusted R 2 = 0.793, p < 0.001 and mean vapor pressure adjusted R 2 = 0.929, p < 0.001 , which linearly associated with mean temperature, and rainfall adjusted R 2 = 0.278, p < 0.001 , which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature coefficient = 0.259 in the current temperature model p = 0.002 , coefficient = 0.328 in mean temperature in the preceding month model p < 0.001 . Conversely, HBoV1 prevalence was negatively correlated with relative humidity coefficient = − 0.126 in the current temperature model p = 0.024 , coefficient = − 0.083 in the temperature delay model p = 0.039 Table 2 . ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports . Overall, 49.2% 5606/11399 of patients tested positive for one or more respiratory pathogens, 2.2% 248/11399 of patients were tested with HBoV1 infection Table 1 . A higher prevalence of HBoV1 was detected in male patients compared with female patients p = 0.019 , consistent with previous reports . Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 . This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference Fig. 2 . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages p < 0.001 . The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period Fig. 3 . In this study, two HBoV1 peaks were observed Fig. 2 . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring , as well as from tropical regions, such as India, where no obvious epidemic season has been found . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity Table 2 . In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study p > 0.05 Table 2 . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections.
What are the symptoms of HBoV1 infection?	cough, rhinitis, fever	[ "BACKGROUND: Human bocavirus 1 HBoV1 is an important cause of acute respiratory illness ARI , yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients ≤14 years old , with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection.", "RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients 45.2%, 112/248 . A significant difference in the prevalence of HBoV1 was found in patients in different age groups p < 0.001 , and the peak prevalence was found in patients aged 7–12 months 4.7%, 56/1203 . Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature.", "Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide.", "Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness ARI , according to different studies . Serological and nucleic acid test results are generally consistent , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms .", "HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications . In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases.", "In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus.", "The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children ≤14 years old hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ .", "This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ . Inclusion criteria were pediatric patients ≤14 years old who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously .", "Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature °C , mean relative humidity % , rainfall mm , mean wind speed m/s , mean air pressure hPa , mean vapor pressure hPa , sunshine duration h . Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit Qiagen, Shanghai, China , respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported .", "Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 SPSS Inc., Chicago, IL, USA . Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016.", "All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 7361:4038 and the median age was 1.75 years interquartile range 0.75-3.83 . Overall, 86.5% 9857/11399 of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 .", "All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 . Two hundred forty-eight of 11,399 patients 2.2% tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 45.2% were co-infected with other pathogens, most frequently with RSV 11.7%, 29/248 Table 1 . The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 .", "The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 . To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups p < 0.001 and the peak prevalence was found in patients aged 7-12 months 4.7%, 56/1203 Fig. 1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1.", "1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′ , has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C mean ± standard deviation , humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 .", "Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure adjusted R 2 = 0.793, p < 0.001 and mean vapor pressure adjusted R 2 = 0.929, p < 0.001 , which linearly associated with mean temperature, and rainfall adjusted R 2 = 0.278, p < 0.001 , which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively.", "The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature coefficient = 0.259 in the current temperature model p = 0.002 , coefficient = 0.328 in mean temperature in the preceding month model p < 0.001 . Conversely, HBoV1 prevalence was negatively correlated with relative humidity coefficient = − 0.126 in the current temperature model p = 0.024 , coefficient = − 0.083 in the temperature delay model p = 0.039 Table 2 . ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 .", "ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports .", "To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports . Overall, 49.2% 5606/11399 of patients tested positive for one or more respiratory pathogens, 2.2% 248/11399 of patients were tested with HBoV1 infection Table 1 . A higher prevalence of HBoV1 was detected in male patients compared with female patients p = 0.019 , consistent with previous reports . Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 .", "Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 . This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference Fig. 2 . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this.", "Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages p < 0.001 . The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 .", "The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region.", "Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period Fig. 3 . In this study, two HBoV1 peaks were observed Fig. 2 . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 .", "The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring , as well as from tropical regions, such as India, where no obvious epidemic season has been found . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model.", "To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity Table 2 . In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 .", "Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 .", "It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study p > 0.05 Table 2 . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation.", "First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections." ]	1,573	3,271	BACKGROUND: Human bocavirus 1 HBoV1 is an important cause of acute respiratory illness ARI , yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients ≤14 years old , with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients 45.2%, 112/248 . A significant difference in the prevalence of HBoV1 was found in patients in different age groups p < 0.001 , and the peak prevalence was found in patients aged 7–12 months 4.7%, 56/1203 . Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness ARI , according to different studies . Serological and nucleic acid test results are generally consistent , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications . In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children ≤14 years old hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ . Inclusion criteria were pediatric patients ≤14 years old who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature °C , mean relative humidity % , rainfall mm , mean wind speed m/s , mean air pressure hPa , mean vapor pressure hPa , sunshine duration h . Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit Qiagen, Shanghai, China , respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 SPSS Inc., Chicago, IL, USA . Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 7361:4038 and the median age was 1.75 years interquartile range 0.75-3.83 . Overall, 86.5% 9857/11399 of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 . Two hundred forty-eight of 11,399 patients 2.2% tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 45.2% were co-infected with other pathogens, most frequently with RSV 11.7%, 29/248 Table 1 . The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 . To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups p < 0.001 and the peak prevalence was found in patients aged 7-12 months 4.7%, 56/1203 Fig. 1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′ , has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C mean ± standard deviation , humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure adjusted R 2 = 0.793, p < 0.001 and mean vapor pressure adjusted R 2 = 0.929, p < 0.001 , which linearly associated with mean temperature, and rainfall adjusted R 2 = 0.278, p < 0.001 , which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature coefficient = 0.259 in the current temperature model p = 0.002 , coefficient = 0.328 in mean temperature in the preceding month model p < 0.001 . Conversely, HBoV1 prevalence was negatively correlated with relative humidity coefficient = − 0.126 in the current temperature model p = 0.024 , coefficient = − 0.083 in the temperature delay model p = 0.039 Table 2 . ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports . Overall, 49.2% 5606/11399 of patients tested positive for one or more respiratory pathogens, 2.2% 248/11399 of patients were tested with HBoV1 infection Table 1 . A higher prevalence of HBoV1 was detected in male patients compared with female patients p = 0.019 , consistent with previous reports . Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 . This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference Fig. 2 . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages p < 0.001 . The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period Fig. 3 . In this study, two HBoV1 peaks were observed Fig. 2 . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring , as well as from tropical regions, such as India, where no obvious epidemic season has been found . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity Table 2 . In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study p > 0.05 Table 2 . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections.
What are the ages of the patients in this study?	≤14 years old	[ "BACKGROUND: Human bocavirus 1 HBoV1 is an important cause of acute respiratory illness ARI , yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients ≤14 years old , with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection.", "RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients 45.2%, 112/248 . A significant difference in the prevalence of HBoV1 was found in patients in different age groups p < 0.001 , and the peak prevalence was found in patients aged 7–12 months 4.7%, 56/1203 . Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature.", "Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide.", "Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness ARI , according to different studies . Serological and nucleic acid test results are generally consistent , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms .", "HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications . In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases.", "In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus.", "The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children ≤14 years old hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ .", "This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ . Inclusion criteria were pediatric patients ≤14 years old who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously .", "Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature °C , mean relative humidity % , rainfall mm , mean wind speed m/s , mean air pressure hPa , mean vapor pressure hPa , sunshine duration h . Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit Qiagen, Shanghai, China , respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported .", "Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 SPSS Inc., Chicago, IL, USA . Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016.", "All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 7361:4038 and the median age was 1.75 years interquartile range 0.75-3.83 . Overall, 86.5% 9857/11399 of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 .", "All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 . Two hundred forty-eight of 11,399 patients 2.2% tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 45.2% were co-infected with other pathogens, most frequently with RSV 11.7%, 29/248 Table 1 . The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 .", "The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 . To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups p < 0.001 and the peak prevalence was found in patients aged 7-12 months 4.7%, 56/1203 Fig. 1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1.", "1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′ , has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C mean ± standard deviation , humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 .", "Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure adjusted R 2 = 0.793, p < 0.001 and mean vapor pressure adjusted R 2 = 0.929, p < 0.001 , which linearly associated with mean temperature, and rainfall adjusted R 2 = 0.278, p < 0.001 , which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively.", "The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature coefficient = 0.259 in the current temperature model p = 0.002 , coefficient = 0.328 in mean temperature in the preceding month model p < 0.001 . Conversely, HBoV1 prevalence was negatively correlated with relative humidity coefficient = − 0.126 in the current temperature model p = 0.024 , coefficient = − 0.083 in the temperature delay model p = 0.039 Table 2 . ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 .", "ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports .", "To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports . Overall, 49.2% 5606/11399 of patients tested positive for one or more respiratory pathogens, 2.2% 248/11399 of patients were tested with HBoV1 infection Table 1 . A higher prevalence of HBoV1 was detected in male patients compared with female patients p = 0.019 , consistent with previous reports . Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 .", "Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 . This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference Fig. 2 . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this.", "Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages p < 0.001 . The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 .", "The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region.", "Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period Fig. 3 . In this study, two HBoV1 peaks were observed Fig. 2 . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 .", "The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring , as well as from tropical regions, such as India, where no obvious epidemic season has been found . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model.", "To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity Table 2 . In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 .", "Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 .", "It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study p > 0.05 Table 2 . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation.", "First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections." ]	1,573	3,272	BACKGROUND: Human bocavirus 1 HBoV1 is an important cause of acute respiratory illness ARI , yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients ≤14 years old , with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients 45.2%, 112/248 . A significant difference in the prevalence of HBoV1 was found in patients in different age groups p < 0.001 , and the peak prevalence was found in patients aged 7–12 months 4.7%, 56/1203 . Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness ARI , according to different studies . Serological and nucleic acid test results are generally consistent , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications . In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children ≤14 years old hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ . Inclusion criteria were pediatric patients ≤14 years old who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature °C , mean relative humidity % , rainfall mm , mean wind speed m/s , mean air pressure hPa , mean vapor pressure hPa , sunshine duration h . Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit Qiagen, Shanghai, China , respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 SPSS Inc., Chicago, IL, USA . Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 7361:4038 and the median age was 1.75 years interquartile range 0.75-3.83 . Overall, 86.5% 9857/11399 of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 . Two hundred forty-eight of 11,399 patients 2.2% tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 45.2% were co-infected with other pathogens, most frequently with RSV 11.7%, 29/248 Table 1 . The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 . To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups p < 0.001 and the peak prevalence was found in patients aged 7-12 months 4.7%, 56/1203 Fig. 1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′ , has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C mean ± standard deviation , humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure adjusted R 2 = 0.793, p < 0.001 and mean vapor pressure adjusted R 2 = 0.929, p < 0.001 , which linearly associated with mean temperature, and rainfall adjusted R 2 = 0.278, p < 0.001 , which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature coefficient = 0.259 in the current temperature model p = 0.002 , coefficient = 0.328 in mean temperature in the preceding month model p < 0.001 . Conversely, HBoV1 prevalence was negatively correlated with relative humidity coefficient = − 0.126 in the current temperature model p = 0.024 , coefficient = − 0.083 in the temperature delay model p = 0.039 Table 2 . ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports . Overall, 49.2% 5606/11399 of patients tested positive for one or more respiratory pathogens, 2.2% 248/11399 of patients were tested with HBoV1 infection Table 1 . A higher prevalence of HBoV1 was detected in male patients compared with female patients p = 0.019 , consistent with previous reports . Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 . This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference Fig. 2 . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages p < 0.001 . The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period Fig. 3 . In this study, two HBoV1 peaks were observed Fig. 2 . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring , as well as from tropical regions, such as India, where no obvious epidemic season has been found . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity Table 2 . In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study p > 0.05 Table 2 . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections.
What was the male to female ratio for this study?	1.82:1	[ "BACKGROUND: Human bocavirus 1 HBoV1 is an important cause of acute respiratory illness ARI , yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients ≤14 years old , with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection.", "RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients 45.2%, 112/248 . A significant difference in the prevalence of HBoV1 was found in patients in different age groups p < 0.001 , and the peak prevalence was found in patients aged 7–12 months 4.7%, 56/1203 . Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature.", "Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide.", "Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness ARI , according to different studies . Serological and nucleic acid test results are generally consistent , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms .", "HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications . In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases.", "In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus.", "The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children ≤14 years old hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ .", "This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ . Inclusion criteria were pediatric patients ≤14 years old who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously .", "Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature °C , mean relative humidity % , rainfall mm , mean wind speed m/s , mean air pressure hPa , mean vapor pressure hPa , sunshine duration h . Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit Qiagen, Shanghai, China , respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported .", "Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 SPSS Inc., Chicago, IL, USA . Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016.", "All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 7361:4038 and the median age was 1.75 years interquartile range 0.75-3.83 . Overall, 86.5% 9857/11399 of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 .", "All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 . Two hundred forty-eight of 11,399 patients 2.2% tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 45.2% were co-infected with other pathogens, most frequently with RSV 11.7%, 29/248 Table 1 . The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 .", "The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 . To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups p < 0.001 and the peak prevalence was found in patients aged 7-12 months 4.7%, 56/1203 Fig. 1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1.", "1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′ , has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C mean ± standard deviation , humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 .", "Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure adjusted R 2 = 0.793, p < 0.001 and mean vapor pressure adjusted R 2 = 0.929, p < 0.001 , which linearly associated with mean temperature, and rainfall adjusted R 2 = 0.278, p < 0.001 , which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively.", "The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature coefficient = 0.259 in the current temperature model p = 0.002 , coefficient = 0.328 in mean temperature in the preceding month model p < 0.001 . Conversely, HBoV1 prevalence was negatively correlated with relative humidity coefficient = − 0.126 in the current temperature model p = 0.024 , coefficient = − 0.083 in the temperature delay model p = 0.039 Table 2 . ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 .", "ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports .", "To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports . Overall, 49.2% 5606/11399 of patients tested positive for one or more respiratory pathogens, 2.2% 248/11399 of patients were tested with HBoV1 infection Table 1 . A higher prevalence of HBoV1 was detected in male patients compared with female patients p = 0.019 , consistent with previous reports . Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 .", "Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 . This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference Fig. 2 . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this.", "Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages p < 0.001 . The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 .", "The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region.", "Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period Fig. 3 . In this study, two HBoV1 peaks were observed Fig. 2 . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 .", "The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring , as well as from tropical regions, such as India, where no obvious epidemic season has been found . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model.", "To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity Table 2 . In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 .", "Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 .", "It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study p > 0.05 Table 2 . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation.", "First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections." ]	1,573	3,273	BACKGROUND: Human bocavirus 1 HBoV1 is an important cause of acute respiratory illness ARI , yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients ≤14 years old , with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients 45.2%, 112/248 . A significant difference in the prevalence of HBoV1 was found in patients in different age groups p < 0.001 , and the peak prevalence was found in patients aged 7–12 months 4.7%, 56/1203 . Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness ARI , according to different studies . Serological and nucleic acid test results are generally consistent , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications . In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children ≤14 years old hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ . Inclusion criteria were pediatric patients ≤14 years old who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature °C , mean relative humidity % , rainfall mm , mean wind speed m/s , mean air pressure hPa , mean vapor pressure hPa , sunshine duration h . Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit Qiagen, Shanghai, China , respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 SPSS Inc., Chicago, IL, USA . Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 7361:4038 and the median age was 1.75 years interquartile range 0.75-3.83 . Overall, 86.5% 9857/11399 of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 . Two hundred forty-eight of 11,399 patients 2.2% tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 45.2% were co-infected with other pathogens, most frequently with RSV 11.7%, 29/248 Table 1 . The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 . To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups p < 0.001 and the peak prevalence was found in patients aged 7-12 months 4.7%, 56/1203 Fig. 1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′ , has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C mean ± standard deviation , humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure adjusted R 2 = 0.793, p < 0.001 and mean vapor pressure adjusted R 2 = 0.929, p < 0.001 , which linearly associated with mean temperature, and rainfall adjusted R 2 = 0.278, p < 0.001 , which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature coefficient = 0.259 in the current temperature model p = 0.002 , coefficient = 0.328 in mean temperature in the preceding month model p < 0.001 . Conversely, HBoV1 prevalence was negatively correlated with relative humidity coefficient = − 0.126 in the current temperature model p = 0.024 , coefficient = − 0.083 in the temperature delay model p = 0.039 Table 2 . ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports . Overall, 49.2% 5606/11399 of patients tested positive for one or more respiratory pathogens, 2.2% 248/11399 of patients were tested with HBoV1 infection Table 1 . A higher prevalence of HBoV1 was detected in male patients compared with female patients p = 0.019 , consistent with previous reports . Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 . This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference Fig. 2 . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages p < 0.001 . The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period Fig. 3 . In this study, two HBoV1 peaks were observed Fig. 2 . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring , as well as from tropical regions, such as India, where no obvious epidemic season has been found . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity Table 2 . In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study p > 0.05 Table 2 . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections.
What health regulations were changes due to the outbreak of C. burnetti?	forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos	[ "BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii .", "To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii . RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away.", "The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters.", "The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3 rd trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii C. burnetii , a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle.", "C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats . High concentrations of C. burnetii can be found in birth products of infected mammals . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances .", "Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache.", "The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis . Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation.", "Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay IFA is considered to be the reference method, complement fixation CF , ELISA and microagglutination MA can also be used . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively .", "Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute.", "Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus.", "Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens ELISA or IFA , a 4-fold increase in anti-phase II IgG antibody titer ELISA or IFA or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors.", "Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case CCS1 case as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing.", "Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 AFE as OR-1 /OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context \"notified case\" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized.", "A case in this context \"notified case\" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition as used in the vendors' study s.b. identified through random sampling of the Soest population within CCS2 s.b. as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day.", "as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below \"Estimation of the overall attack rate\" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. .", "Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below \"Estimation of the overall attack rate\" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. . In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires.", "In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units.", "Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose 360° in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east Figure 1 . For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e.", "In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen in stand units or meters and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms.", "We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition as defined in the cohort study on vendors . The overall attack rate among adults was estimated based on the following sources: . Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later \"ill with fever\"; .", "Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later \"ill with fever\"; . Results of the small cohort of 9 sailor friends s.a. ; . Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever s.a. \"determination of outbreak size and descriptive epidemiology\" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source.", "Local health departments that identified outbreak cases of Q fever s.a. \"determination of outbreak size and descriptive epidemiology\" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in ., . and . we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors.", "we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children numerator . For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported.", "For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions number of visitors, proportion of adult visitors and clinical attack rate among adults we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks 70 animals . Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture.", "Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 CDC, Atlanta, USA . Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed.", "P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends.", "It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents.", "Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. Figure 2 . If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases mean +/-2 standard deviations had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female.", "The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female. Of the notified cases, 189 63% were from the county of Soest, 104 35% were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state Northrhine Westphalia and 6 2% were from five other federal states in Germany Figure 3 . Only eight 3% cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively Figure 4 . 75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized.", "75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 56% outbreak cases; 66 22% were confirmed by an increase in anti-phase II antibody titer CF , 89 30% had IgM antibodies against phase II antigens, 11 4% were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 44% cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever.", "18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two 11% of 18 cardiological patients and four 36% of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman case 3; Table 1 . Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections.", "Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23 . Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 .", "Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 . Touching the sheep was also significantly more common among cases 5/20 25% CCS2 cases vs. 0/22 0% controls; OR undefined; lower 95% CI = 1.1; p = 0.02 . 17 85% of 20 CCS2 cases, but only 6 25% of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was \"having passed by the pen without stopping\" OR = 17.0; 95%CI = 3.0-112.5; p < 0.01 . Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% .", "Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% . Fourty-five 56% were male and 35 44% were female. 13 16% met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2.", "Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen section 4, Figure 1 , 4 9% of 44 vendors became cases, compared to 2 13% of 15 persons who worked in section 1 p = 0.6 . Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 32% became cases. Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 .", "Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 . In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town.", "The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 25% tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise.", "The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market . More recently a large outbreak occurred in Jena Thuringia in 2005 with 322 reported cases associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately.", "The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature .", "The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature . An additional interview with the two cases with early onset 2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for \"number of visitors\", \"proportion of adult visitors\" and \"attack rate among adults\" see Table 3 . Displayed are attack rates and 95% confidence intervals.", "Scenarios 1-8 varied in the assumptions made for \"number of visitors\", \"proportion of adult visitors\" and \"attack rate among adults\" see Table 3 . Displayed are attack rates and 95% confidence intervals. respectively could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever.", "The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 .", "According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen.", "Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep.", "The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep n = 1000-2000 the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided .", "In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic 50% 3/6 is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system.", "In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults.", "Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks .", "Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that in such a setting can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a \"deprived\" urban population \"in touch\" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, .", "During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a \"deprived\" urban population \"in touch\" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals.", "Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author s declare that they have no competing interests." ]	1,583	5,201	BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii . RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3 rd trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii C. burnetii , a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats . High concentrations of C. burnetii can be found in birth products of infected mammals . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis . Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay IFA is considered to be the reference method, complement fixation CF , ELISA and microagglutination MA can also be used . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens ELISA or IFA , a 4-fold increase in anti-phase II IgG antibody titer ELISA or IFA or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case CCS1 case as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 AFE as OR-1 /OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context "notified case" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition as used in the vendors' study s.b. identified through random sampling of the Soest population within CCS2 s.b. as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below "Estimation of the overall attack rate" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. . In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose 360° in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east Figure 1 . For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen in stand units or meters and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition as defined in the cohort study on vendors . The overall attack rate among adults was estimated based on the following sources: . Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later "ill with fever"; . Results of the small cohort of 9 sailor friends s.a. ; . Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever s.a. "determination of outbreak size and descriptive epidemiology" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in ., . and . we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children numerator . For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions number of visitors, proportion of adult visitors and clinical attack rate among adults we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks 70 animals . Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 CDC, Atlanta, USA . Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. Figure 2 . If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases mean +/-2 standard deviations had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female. Of the notified cases, 189 63% were from the county of Soest, 104 35% were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state Northrhine Westphalia and 6 2% were from five other federal states in Germany Figure 3 . Only eight 3% cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively Figure 4 . 75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 56% outbreak cases; 66 22% were confirmed by an increase in anti-phase II antibody titer CF , 89 30% had IgM antibodies against phase II antigens, 11 4% were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 44% cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two 11% of 18 cardiological patients and four 36% of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman case 3; Table 1 . Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23 . Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 . Touching the sheep was also significantly more common among cases 5/20 25% CCS2 cases vs. 0/22 0% controls; OR undefined; lower 95% CI = 1.1; p = 0.02 . 17 85% of 20 CCS2 cases, but only 6 25% of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was "having passed by the pen without stopping" OR = 17.0; 95%CI = 3.0-112.5; p < 0.01 . Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% . Fourty-five 56% were male and 35 44% were female. 13 16% met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen section 4, Figure 1 , 4 9% of 44 vendors became cases, compared to 2 13% of 15 persons who worked in section 1 p = 0.6 . Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 32% became cases. Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 . In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 25% tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market . More recently a large outbreak occurred in Jena Thuringia in 2005 with 322 reported cases associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature . An additional interview with the two cases with early onset 2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for "number of visitors", "proportion of adult visitors" and "attack rate among adults" see Table 3 . Displayed are attack rates and 95% confidence intervals. respectively could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep n = 1000-2000 the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic 50% 3/6 is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that in such a setting can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a "deprived" urban population "in touch" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author s declare that they have no competing interests.
What was the median seropresence of C. burnetti in sheep flocks not linked to human outbreaks?	1%	[ "BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii .", "To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii . RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away.", "The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters.", "The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3 rd trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii C. burnetii , a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle.", "C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats . High concentrations of C. burnetii can be found in birth products of infected mammals . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances .", "Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache.", "The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis . Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation.", "Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay IFA is considered to be the reference method, complement fixation CF , ELISA and microagglutination MA can also be used . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively .", "Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute.", "Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus.", "Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens ELISA or IFA , a 4-fold increase in anti-phase II IgG antibody titer ELISA or IFA or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors.", "Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case CCS1 case as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing.", "Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 AFE as OR-1 /OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context \"notified case\" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized.", "A case in this context \"notified case\" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition as used in the vendors' study s.b. identified through random sampling of the Soest population within CCS2 s.b. as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day.", "as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below \"Estimation of the overall attack rate\" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. .", "Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below \"Estimation of the overall attack rate\" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. . In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires.", "In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units.", "Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose 360° in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east Figure 1 . For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e.", "In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen in stand units or meters and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms.", "We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition as defined in the cohort study on vendors . The overall attack rate among adults was estimated based on the following sources: . Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later \"ill with fever\"; .", "Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later \"ill with fever\"; . Results of the small cohort of 9 sailor friends s.a. ; . Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever s.a. \"determination of outbreak size and descriptive epidemiology\" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source.", "Local health departments that identified outbreak cases of Q fever s.a. \"determination of outbreak size and descriptive epidemiology\" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in ., . and . we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors.", "we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children numerator . For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported.", "For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions number of visitors, proportion of adult visitors and clinical attack rate among adults we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks 70 animals . Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture.", "Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 CDC, Atlanta, USA . Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed.", "P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends.", "It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents.", "Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. Figure 2 . If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases mean +/-2 standard deviations had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female.", "The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female. Of the notified cases, 189 63% were from the county of Soest, 104 35% were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state Northrhine Westphalia and 6 2% were from five other federal states in Germany Figure 3 . Only eight 3% cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively Figure 4 . 75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized.", "75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 56% outbreak cases; 66 22% were confirmed by an increase in anti-phase II antibody titer CF , 89 30% had IgM antibodies against phase II antigens, 11 4% were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 44% cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever.", "18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two 11% of 18 cardiological patients and four 36% of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman case 3; Table 1 . Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections.", "Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23 . Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 .", "Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 . Touching the sheep was also significantly more common among cases 5/20 25% CCS2 cases vs. 0/22 0% controls; OR undefined; lower 95% CI = 1.1; p = 0.02 . 17 85% of 20 CCS2 cases, but only 6 25% of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was \"having passed by the pen without stopping\" OR = 17.0; 95%CI = 3.0-112.5; p < 0.01 . Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% .", "Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% . Fourty-five 56% were male and 35 44% were female. 13 16% met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2.", "Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen section 4, Figure 1 , 4 9% of 44 vendors became cases, compared to 2 13% of 15 persons who worked in section 1 p = 0.6 . Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 32% became cases. Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 .", "Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 . In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town.", "The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 25% tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise.", "The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market . More recently a large outbreak occurred in Jena Thuringia in 2005 with 322 reported cases associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately.", "The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature .", "The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature . An additional interview with the two cases with early onset 2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for \"number of visitors\", \"proportion of adult visitors\" and \"attack rate among adults\" see Table 3 . Displayed are attack rates and 95% confidence intervals.", "Scenarios 1-8 varied in the assumptions made for \"number of visitors\", \"proportion of adult visitors\" and \"attack rate among adults\" see Table 3 . Displayed are attack rates and 95% confidence intervals. respectively could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever.", "The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 .", "According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen.", "Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep.", "The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep n = 1000-2000 the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided .", "In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic 50% 3/6 is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system.", "In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults.", "Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks .", "Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that in such a setting can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a \"deprived\" urban population \"in touch\" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, .", "During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a \"deprived\" urban population \"in touch\" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals.", "Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author s declare that they have no competing interests." ]	1,583	5,202	BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii . RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3 rd trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii C. burnetii , a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats . High concentrations of C. burnetii can be found in birth products of infected mammals . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis . Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay IFA is considered to be the reference method, complement fixation CF , ELISA and microagglutination MA can also be used . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens ELISA or IFA , a 4-fold increase in anti-phase II IgG antibody titer ELISA or IFA or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case CCS1 case as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 AFE as OR-1 /OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context "notified case" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition as used in the vendors' study s.b. identified through random sampling of the Soest population within CCS2 s.b. as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below "Estimation of the overall attack rate" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. . In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose 360° in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east Figure 1 . For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen in stand units or meters and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition as defined in the cohort study on vendors . The overall attack rate among adults was estimated based on the following sources: . Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later "ill with fever"; . Results of the small cohort of 9 sailor friends s.a. ; . Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever s.a. "determination of outbreak size and descriptive epidemiology" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in ., . and . we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children numerator . For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions number of visitors, proportion of adult visitors and clinical attack rate among adults we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks 70 animals . Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 CDC, Atlanta, USA . Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. Figure 2 . If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases mean +/-2 standard deviations had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female. Of the notified cases, 189 63% were from the county of Soest, 104 35% were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state Northrhine Westphalia and 6 2% were from five other federal states in Germany Figure 3 . Only eight 3% cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively Figure 4 . 75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 56% outbreak cases; 66 22% were confirmed by an increase in anti-phase II antibody titer CF , 89 30% had IgM antibodies against phase II antigens, 11 4% were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 44% cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two 11% of 18 cardiological patients and four 36% of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman case 3; Table 1 . Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23 . Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 . Touching the sheep was also significantly more common among cases 5/20 25% CCS2 cases vs. 0/22 0% controls; OR undefined; lower 95% CI = 1.1; p = 0.02 . 17 85% of 20 CCS2 cases, but only 6 25% of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was "having passed by the pen without stopping" OR = 17.0; 95%CI = 3.0-112.5; p < 0.01 . Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% . Fourty-five 56% were male and 35 44% were female. 13 16% met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen section 4, Figure 1 , 4 9% of 44 vendors became cases, compared to 2 13% of 15 persons who worked in section 1 p = 0.6 . Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 32% became cases. Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 . In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 25% tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market . More recently a large outbreak occurred in Jena Thuringia in 2005 with 322 reported cases associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature . An additional interview with the two cases with early onset 2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for "number of visitors", "proportion of adult visitors" and "attack rate among adults" see Table 3 . Displayed are attack rates and 95% confidence intervals. respectively could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep n = 1000-2000 the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic 50% 3/6 is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that in such a setting can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a "deprived" urban population "in touch" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author s declare that they have no competing interests.
What important risk factors to infection were found during the second case-controlled study?	close proximity to the ewe and duration of exposure	[ "BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii .", "To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii . RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away.", "The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters.", "The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3 rd trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii C. burnetii , a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle.", "C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats . High concentrations of C. burnetii can be found in birth products of infected mammals . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances .", "Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache.", "The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis . Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation.", "Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay IFA is considered to be the reference method, complement fixation CF , ELISA and microagglutination MA can also be used . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively .", "Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute.", "Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus.", "Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens ELISA or IFA , a 4-fold increase in anti-phase II IgG antibody titer ELISA or IFA or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors.", "Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case CCS1 case as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing.", "Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 AFE as OR-1 /OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context \"notified case\" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized.", "A case in this context \"notified case\" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition as used in the vendors' study s.b. identified through random sampling of the Soest population within CCS2 s.b. as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day.", "as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below \"Estimation of the overall attack rate\" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. .", "Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below \"Estimation of the overall attack rate\" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. . In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires.", "In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units.", "Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose 360° in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east Figure 1 . For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e.", "In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen in stand units or meters and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms.", "We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition as defined in the cohort study on vendors . The overall attack rate among adults was estimated based on the following sources: . Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later \"ill with fever\"; .", "Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later \"ill with fever\"; . Results of the small cohort of 9 sailor friends s.a. ; . Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever s.a. \"determination of outbreak size and descriptive epidemiology\" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source.", "Local health departments that identified outbreak cases of Q fever s.a. \"determination of outbreak size and descriptive epidemiology\" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in ., . and . we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors.", "we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children numerator . For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported.", "For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions number of visitors, proportion of adult visitors and clinical attack rate among adults we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks 70 animals . Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture.", "Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 CDC, Atlanta, USA . Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed.", "P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends.", "It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents.", "Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. Figure 2 . If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases mean +/-2 standard deviations had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female.", "The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female. Of the notified cases, 189 63% were from the county of Soest, 104 35% were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state Northrhine Westphalia and 6 2% were from five other federal states in Germany Figure 3 . Only eight 3% cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively Figure 4 . 75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized.", "75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 56% outbreak cases; 66 22% were confirmed by an increase in anti-phase II antibody titer CF , 89 30% had IgM antibodies against phase II antigens, 11 4% were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 44% cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever.", "18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two 11% of 18 cardiological patients and four 36% of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman case 3; Table 1 . Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections.", "Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23 . Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 .", "Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 . Touching the sheep was also significantly more common among cases 5/20 25% CCS2 cases vs. 0/22 0% controls; OR undefined; lower 95% CI = 1.1; p = 0.02 . 17 85% of 20 CCS2 cases, but only 6 25% of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was \"having passed by the pen without stopping\" OR = 17.0; 95%CI = 3.0-112.5; p < 0.01 . Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% .", "Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% . Fourty-five 56% were male and 35 44% were female. 13 16% met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2.", "Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen section 4, Figure 1 , 4 9% of 44 vendors became cases, compared to 2 13% of 15 persons who worked in section 1 p = 0.6 . Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 32% became cases. Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 .", "Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 . In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town.", "The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 25% tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise.", "The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market . More recently a large outbreak occurred in Jena Thuringia in 2005 with 322 reported cases associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately.", "The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature .", "The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature . An additional interview with the two cases with early onset 2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for \"number of visitors\", \"proportion of adult visitors\" and \"attack rate among adults\" see Table 3 . Displayed are attack rates and 95% confidence intervals.", "Scenarios 1-8 varied in the assumptions made for \"number of visitors\", \"proportion of adult visitors\" and \"attack rate among adults\" see Table 3 . Displayed are attack rates and 95% confidence intervals. respectively could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever.", "The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 .", "According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen.", "Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep.", "The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep n = 1000-2000 the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided .", "In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic 50% 3/6 is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system.", "In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults.", "Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks .", "Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that in such a setting can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a \"deprived\" urban population \"in touch\" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, .", "During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a \"deprived\" urban population \"in touch\" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals.", "Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author s declare that they have no competing interests." ]	1,583	5,203	BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii . RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3 rd trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii C. burnetii , a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats . High concentrations of C. burnetii can be found in birth products of infected mammals . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis . Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay IFA is considered to be the reference method, complement fixation CF , ELISA and microagglutination MA can also be used . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens ELISA or IFA , a 4-fold increase in anti-phase II IgG antibody titer ELISA or IFA or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case CCS1 case as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 AFE as OR-1 /OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context "notified case" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition as used in the vendors' study s.b. identified through random sampling of the Soest population within CCS2 s.b. as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below "Estimation of the overall attack rate" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. . In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose 360° in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east Figure 1 . For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen in stand units or meters and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition as defined in the cohort study on vendors . The overall attack rate among adults was estimated based on the following sources: . Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later "ill with fever"; . Results of the small cohort of 9 sailor friends s.a. ; . Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever s.a. "determination of outbreak size and descriptive epidemiology" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in ., . and . we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children numerator . For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions number of visitors, proportion of adult visitors and clinical attack rate among adults we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks 70 animals . Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 CDC, Atlanta, USA . Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. Figure 2 . If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases mean +/-2 standard deviations had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female. Of the notified cases, 189 63% were from the county of Soest, 104 35% were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state Northrhine Westphalia and 6 2% were from five other federal states in Germany Figure 3 . Only eight 3% cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively Figure 4 . 75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 56% outbreak cases; 66 22% were confirmed by an increase in anti-phase II antibody titer CF , 89 30% had IgM antibodies against phase II antigens, 11 4% were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 44% cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two 11% of 18 cardiological patients and four 36% of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman case 3; Table 1 . Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23 . Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 . Touching the sheep was also significantly more common among cases 5/20 25% CCS2 cases vs. 0/22 0% controls; OR undefined; lower 95% CI = 1.1; p = 0.02 . 17 85% of 20 CCS2 cases, but only 6 25% of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was "having passed by the pen without stopping" OR = 17.0; 95%CI = 3.0-112.5; p < 0.01 . Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% . Fourty-five 56% were male and 35 44% were female. 13 16% met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen section 4, Figure 1 , 4 9% of 44 vendors became cases, compared to 2 13% of 15 persons who worked in section 1 p = 0.6 . Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 32% became cases. Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 . In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 25% tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market . More recently a large outbreak occurred in Jena Thuringia in 2005 with 322 reported cases associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature . An additional interview with the two cases with early onset 2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for "number of visitors", "proportion of adult visitors" and "attack rate among adults" see Table 3 . Displayed are attack rates and 95% confidence intervals. respectively could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep n = 1000-2000 the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic 50% 3/6 is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that in such a setting can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a "deprived" urban population "in touch" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author s declare that they have no competing interests.
What was the interquartile range of the incubation period?	16 to 24 days	[ "BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii .", "To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii . RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away.", "The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters.", "The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3 rd trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii C. burnetii , a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle.", "C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats . High concentrations of C. burnetii can be found in birth products of infected mammals . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances .", "Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache.", "The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis . Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation.", "Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay IFA is considered to be the reference method, complement fixation CF , ELISA and microagglutination MA can also be used . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively .", "Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute.", "Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus.", "Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens ELISA or IFA , a 4-fold increase in anti-phase II IgG antibody titer ELISA or IFA or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors.", "Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case CCS1 case as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing.", "Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 AFE as OR-1 /OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context \"notified case\" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized.", "A case in this context \"notified case\" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition as used in the vendors' study s.b. identified through random sampling of the Soest population within CCS2 s.b. as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day.", "as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below \"Estimation of the overall attack rate\" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. .", "Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below \"Estimation of the overall attack rate\" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. . In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires.", "In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units.", "Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose 360° in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east Figure 1 . For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e.", "In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen in stand units or meters and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms.", "We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition as defined in the cohort study on vendors . The overall attack rate among adults was estimated based on the following sources: . Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later \"ill with fever\"; .", "Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later \"ill with fever\"; . Results of the small cohort of 9 sailor friends s.a. ; . Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever s.a. \"determination of outbreak size and descriptive epidemiology\" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source.", "Local health departments that identified outbreak cases of Q fever s.a. \"determination of outbreak size and descriptive epidemiology\" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in ., . and . we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors.", "we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children numerator . For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported.", "For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions number of visitors, proportion of adult visitors and clinical attack rate among adults we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks 70 animals . Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture.", "Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 CDC, Atlanta, USA . Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed.", "P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends.", "It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents.", "Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. Figure 2 . If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases mean +/-2 standard deviations had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female.", "The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female. Of the notified cases, 189 63% were from the county of Soest, 104 35% were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state Northrhine Westphalia and 6 2% were from five other federal states in Germany Figure 3 . Only eight 3% cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively Figure 4 . 75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized.", "75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 56% outbreak cases; 66 22% were confirmed by an increase in anti-phase II antibody titer CF , 89 30% had IgM antibodies against phase II antigens, 11 4% were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 44% cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever.", "18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two 11% of 18 cardiological patients and four 36% of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman case 3; Table 1 . Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections.", "Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23 . Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 .", "Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 . Touching the sheep was also significantly more common among cases 5/20 25% CCS2 cases vs. 0/22 0% controls; OR undefined; lower 95% CI = 1.1; p = 0.02 . 17 85% of 20 CCS2 cases, but only 6 25% of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was \"having passed by the pen without stopping\" OR = 17.0; 95%CI = 3.0-112.5; p < 0.01 . Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% .", "Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% . Fourty-five 56% were male and 35 44% were female. 13 16% met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2.", "Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen section 4, Figure 1 , 4 9% of 44 vendors became cases, compared to 2 13% of 15 persons who worked in section 1 p = 0.6 . Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 32% became cases. Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 .", "Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 . In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town.", "The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 25% tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise.", "The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market . More recently a large outbreak occurred in Jena Thuringia in 2005 with 322 reported cases associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately.", "The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature .", "The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature . An additional interview with the two cases with early onset 2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for \"number of visitors\", \"proportion of adult visitors\" and \"attack rate among adults\" see Table 3 . Displayed are attack rates and 95% confidence intervals.", "Scenarios 1-8 varied in the assumptions made for \"number of visitors\", \"proportion of adult visitors\" and \"attack rate among adults\" see Table 3 . Displayed are attack rates and 95% confidence intervals. respectively could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever.", "The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 .", "According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen.", "Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep.", "The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep n = 1000-2000 the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided .", "In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic 50% 3/6 is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system.", "In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults.", "Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks .", "Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that in such a setting can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a \"deprived\" urban population \"in touch\" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, .", "During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a \"deprived\" urban population \"in touch\" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals.", "Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author s declare that they have no competing interests." ]	1,583	5,204	BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii . RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3 rd trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii C. burnetii , a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats . High concentrations of C. burnetii can be found in birth products of infected mammals . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis . Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay IFA is considered to be the reference method, complement fixation CF , ELISA and microagglutination MA can also be used . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens ELISA or IFA , a 4-fold increase in anti-phase II IgG antibody titer ELISA or IFA or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case CCS1 case as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 AFE as OR-1 /OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context "notified case" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition as used in the vendors' study s.b. identified through random sampling of the Soest population within CCS2 s.b. as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below "Estimation of the overall attack rate" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. . In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose 360° in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east Figure 1 . For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen in stand units or meters and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition as defined in the cohort study on vendors . The overall attack rate among adults was estimated based on the following sources: . Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later "ill with fever"; . Results of the small cohort of 9 sailor friends s.a. ; . Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever s.a. "determination of outbreak size and descriptive epidemiology" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in ., . and . we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children numerator . For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions number of visitors, proportion of adult visitors and clinical attack rate among adults we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks 70 animals . Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 CDC, Atlanta, USA . Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. Figure 2 . If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases mean +/-2 standard deviations had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female. Of the notified cases, 189 63% were from the county of Soest, 104 35% were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state Northrhine Westphalia and 6 2% were from five other federal states in Germany Figure 3 . Only eight 3% cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively Figure 4 . 75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 56% outbreak cases; 66 22% were confirmed by an increase in anti-phase II antibody titer CF , 89 30% had IgM antibodies against phase II antigens, 11 4% were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 44% cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two 11% of 18 cardiological patients and four 36% of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman case 3; Table 1 . Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23 . Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 . Touching the sheep was also significantly more common among cases 5/20 25% CCS2 cases vs. 0/22 0% controls; OR undefined; lower 95% CI = 1.1; p = 0.02 . 17 85% of 20 CCS2 cases, but only 6 25% of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was "having passed by the pen without stopping" OR = 17.0; 95%CI = 3.0-112.5; p < 0.01 . Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% . Fourty-five 56% were male and 35 44% were female. 13 16% met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen section 4, Figure 1 , 4 9% of 44 vendors became cases, compared to 2 13% of 15 persons who worked in section 1 p = 0.6 . Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 32% became cases. Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 . In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 25% tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market . More recently a large outbreak occurred in Jena Thuringia in 2005 with 322 reported cases associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature . An additional interview with the two cases with early onset 2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for "number of visitors", "proportion of adult visitors" and "attack rate among adults" see Table 3 . Displayed are attack rates and 95% confidence intervals. respectively could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep n = 1000-2000 the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic 50% 3/6 is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that in such a setting can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a "deprived" urban population "in touch" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author s declare that they have no competing interests.
How many controls were used in the second case study?	36	[ "BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii .", "To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii . RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away.", "The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters.", "The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3 rd trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii C. burnetii , a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle.", "C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats . High concentrations of C. burnetii can be found in birth products of infected mammals . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances .", "Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache.", "The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis . Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation.", "Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay IFA is considered to be the reference method, complement fixation CF , ELISA and microagglutination MA can also be used . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively .", "Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute.", "Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus.", "Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens ELISA or IFA , a 4-fold increase in anti-phase II IgG antibody titer ELISA or IFA or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors.", "Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case CCS1 case as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing.", "Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 AFE as OR-1 /OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context \"notified case\" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized.", "A case in this context \"notified case\" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition as used in the vendors' study s.b. identified through random sampling of the Soest population within CCS2 s.b. as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day.", "as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below \"Estimation of the overall attack rate\" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. .", "Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below \"Estimation of the overall attack rate\" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. . In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires.", "In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units.", "Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose 360° in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east Figure 1 . For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e.", "In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen in stand units or meters and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms.", "We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition as defined in the cohort study on vendors . The overall attack rate among adults was estimated based on the following sources: . Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later \"ill with fever\"; .", "Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later \"ill with fever\"; . Results of the small cohort of 9 sailor friends s.a. ; . Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever s.a. \"determination of outbreak size and descriptive epidemiology\" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source.", "Local health departments that identified outbreak cases of Q fever s.a. \"determination of outbreak size and descriptive epidemiology\" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in ., . and . we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors.", "we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children numerator . For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported.", "For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions number of visitors, proportion of adult visitors and clinical attack rate among adults we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks 70 animals . Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture.", "Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 CDC, Atlanta, USA . Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed.", "P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends.", "It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents.", "Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. Figure 2 . If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases mean +/-2 standard deviations had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female.", "The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female. Of the notified cases, 189 63% were from the county of Soest, 104 35% were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state Northrhine Westphalia and 6 2% were from five other federal states in Germany Figure 3 . Only eight 3% cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively Figure 4 . 75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized.", "75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 56% outbreak cases; 66 22% were confirmed by an increase in anti-phase II antibody titer CF , 89 30% had IgM antibodies against phase II antigens, 11 4% were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 44% cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever.", "18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two 11% of 18 cardiological patients and four 36% of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman case 3; Table 1 . Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections.", "Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23 . Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 .", "Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 . Touching the sheep was also significantly more common among cases 5/20 25% CCS2 cases vs. 0/22 0% controls; OR undefined; lower 95% CI = 1.1; p = 0.02 . 17 85% of 20 CCS2 cases, but only 6 25% of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was \"having passed by the pen without stopping\" OR = 17.0; 95%CI = 3.0-112.5; p < 0.01 . Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% .", "Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% . Fourty-five 56% were male and 35 44% were female. 13 16% met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2.", "Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen section 4, Figure 1 , 4 9% of 44 vendors became cases, compared to 2 13% of 15 persons who worked in section 1 p = 0.6 . Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 32% became cases. Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 .", "Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 . In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town.", "The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 25% tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise.", "The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market . More recently a large outbreak occurred in Jena Thuringia in 2005 with 322 reported cases associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately.", "The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature .", "The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature . An additional interview with the two cases with early onset 2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for \"number of visitors\", \"proportion of adult visitors\" and \"attack rate among adults\" see Table 3 . Displayed are attack rates and 95% confidence intervals.", "Scenarios 1-8 varied in the assumptions made for \"number of visitors\", \"proportion of adult visitors\" and \"attack rate among adults\" see Table 3 . Displayed are attack rates and 95% confidence intervals. respectively could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever.", "The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 .", "According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen.", "Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep.", "The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep n = 1000-2000 the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided .", "In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic 50% 3/6 is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system.", "In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults.", "Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks .", "Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that in such a setting can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a \"deprived\" urban population \"in touch\" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, .", "During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a \"deprived\" urban population \"in touch\" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals.", "Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author s declare that they have no competing interests." ]	1,583	5,205	BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii . RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3 rd trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii C. burnetii , a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats . High concentrations of C. burnetii can be found in birth products of infected mammals . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis . Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay IFA is considered to be the reference method, complement fixation CF , ELISA and microagglutination MA can also be used . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens ELISA or IFA , a 4-fold increase in anti-phase II IgG antibody titer ELISA or IFA or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case CCS1 case as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 AFE as OR-1 /OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context "notified case" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition as used in the vendors' study s.b. identified through random sampling of the Soest population within CCS2 s.b. as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below "Estimation of the overall attack rate" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. . In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose 360° in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east Figure 1 . For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen in stand units or meters and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition as defined in the cohort study on vendors . The overall attack rate among adults was estimated based on the following sources: . Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later "ill with fever"; . Results of the small cohort of 9 sailor friends s.a. ; . Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever s.a. "determination of outbreak size and descriptive epidemiology" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in ., . and . we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children numerator . For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions number of visitors, proportion of adult visitors and clinical attack rate among adults we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks 70 animals . Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 CDC, Atlanta, USA . Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. Figure 2 . If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases mean +/-2 standard deviations had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female. Of the notified cases, 189 63% were from the county of Soest, 104 35% were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state Northrhine Westphalia and 6 2% were from five other federal states in Germany Figure 3 . Only eight 3% cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively Figure 4 . 75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 56% outbreak cases; 66 22% were confirmed by an increase in anti-phase II antibody titer CF , 89 30% had IgM antibodies against phase II antigens, 11 4% were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 44% cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two 11% of 18 cardiological patients and four 36% of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman case 3; Table 1 . Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23 . Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 . Touching the sheep was also significantly more common among cases 5/20 25% CCS2 cases vs. 0/22 0% controls; OR undefined; lower 95% CI = 1.1; p = 0.02 . 17 85% of 20 CCS2 cases, but only 6 25% of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was "having passed by the pen without stopping" OR = 17.0; 95%CI = 3.0-112.5; p < 0.01 . Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% . Fourty-five 56% were male and 35 44% were female. 13 16% met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen section 4, Figure 1 , 4 9% of 44 vendors became cases, compared to 2 13% of 15 persons who worked in section 1 p = 0.6 . Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 32% became cases. Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 . In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 25% tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market . More recently a large outbreak occurred in Jena Thuringia in 2005 with 322 reported cases associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature . An additional interview with the two cases with early onset 2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for "number of visitors", "proportion of adult visitors" and "attack rate among adults" see Table 3 . Displayed are attack rates and 95% confidence intervals. respectively could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep n = 1000-2000 the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic 50% 3/6 is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that in such a setting can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a "deprived" urban population "in touch" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author s declare that they have no competing interests.
What public event was linked with the outbreak?	farmers' market	[ "BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii .", "To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii . RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away.", "The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters.", "The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3 rd trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii C. burnetii , a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle.", "C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats . High concentrations of C. burnetii can be found in birth products of infected mammals . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances .", "Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache.", "The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis . Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation.", "Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay IFA is considered to be the reference method, complement fixation CF , ELISA and microagglutination MA can also be used . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively .", "Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute.", "Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus.", "Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens ELISA or IFA , a 4-fold increase in anti-phase II IgG antibody titer ELISA or IFA or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors.", "Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case CCS1 case as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing.", "Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 AFE as OR-1 /OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context \"notified case\" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized.", "A case in this context \"notified case\" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition as used in the vendors' study s.b. identified through random sampling of the Soest population within CCS2 s.b. as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day.", "as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below \"Estimation of the overall attack rate\" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. .", "Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below \"Estimation of the overall attack rate\" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. . In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires.", "In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units.", "Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose 360° in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east Figure 1 . For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e.", "In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen in stand units or meters and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms.", "We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition as defined in the cohort study on vendors . The overall attack rate among adults was estimated based on the following sources: . Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later \"ill with fever\"; .", "Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later \"ill with fever\"; . Results of the small cohort of 9 sailor friends s.a. ; . Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever s.a. \"determination of outbreak size and descriptive epidemiology\" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source.", "Local health departments that identified outbreak cases of Q fever s.a. \"determination of outbreak size and descriptive epidemiology\" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in ., . and . we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors.", "we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children numerator . For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported.", "For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions number of visitors, proportion of adult visitors and clinical attack rate among adults we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks 70 animals . Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture.", "Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 CDC, Atlanta, USA . Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed.", "P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends.", "It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents.", "Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. Figure 2 . If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases mean +/-2 standard deviations had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female.", "The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female. Of the notified cases, 189 63% were from the county of Soest, 104 35% were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state Northrhine Westphalia and 6 2% were from five other federal states in Germany Figure 3 . Only eight 3% cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively Figure 4 . 75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized.", "75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 56% outbreak cases; 66 22% were confirmed by an increase in anti-phase II antibody titer CF , 89 30% had IgM antibodies against phase II antigens, 11 4% were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 44% cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever.", "18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two 11% of 18 cardiological patients and four 36% of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman case 3; Table 1 . Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections.", "Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23 . Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 .", "Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 . Touching the sheep was also significantly more common among cases 5/20 25% CCS2 cases vs. 0/22 0% controls; OR undefined; lower 95% CI = 1.1; p = 0.02 . 17 85% of 20 CCS2 cases, but only 6 25% of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was \"having passed by the pen without stopping\" OR = 17.0; 95%CI = 3.0-112.5; p < 0.01 . Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% .", "Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% . Fourty-five 56% were male and 35 44% were female. 13 16% met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2.", "Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen section 4, Figure 1 , 4 9% of 44 vendors became cases, compared to 2 13% of 15 persons who worked in section 1 p = 0.6 . Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 32% became cases. Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 .", "Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 . In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town.", "The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 25% tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise.", "The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market . More recently a large outbreak occurred in Jena Thuringia in 2005 with 322 reported cases associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately.", "The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature .", "The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature . An additional interview with the two cases with early onset 2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for \"number of visitors\", \"proportion of adult visitors\" and \"attack rate among adults\" see Table 3 . Displayed are attack rates and 95% confidence intervals.", "Scenarios 1-8 varied in the assumptions made for \"number of visitors\", \"proportion of adult visitors\" and \"attack rate among adults\" see Table 3 . Displayed are attack rates and 95% confidence intervals. respectively could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever.", "The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 .", "According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen.", "Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep.", "The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep n = 1000-2000 the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided .", "In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic 50% 3/6 is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system.", "In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults.", "Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks .", "Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that in such a setting can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a \"deprived\" urban population \"in touch\" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, .", "During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a \"deprived\" urban population \"in touch\" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals.", "Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author s declare that they have no competing interests." ]	1,583	5,206	BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii . RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3 rd trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii C. burnetii , a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats . High concentrations of C. burnetii can be found in birth products of infected mammals . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis . Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay IFA is considered to be the reference method, complement fixation CF , ELISA and microagglutination MA can also be used . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens ELISA or IFA , a 4-fold increase in anti-phase II IgG antibody titer ELISA or IFA or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case CCS1 case as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 AFE as OR-1 /OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context "notified case" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition as used in the vendors' study s.b. identified through random sampling of the Soest population within CCS2 s.b. as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below "Estimation of the overall attack rate" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. . In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose 360° in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east Figure 1 . For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen in stand units or meters and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition as defined in the cohort study on vendors . The overall attack rate among adults was estimated based on the following sources: . Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later "ill with fever"; . Results of the small cohort of 9 sailor friends s.a. ; . Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever s.a. "determination of outbreak size and descriptive epidemiology" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in ., . and . we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children numerator . For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions number of visitors, proportion of adult visitors and clinical attack rate among adults we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks 70 animals . Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 CDC, Atlanta, USA . Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. Figure 2 . If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases mean +/-2 standard deviations had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female. Of the notified cases, 189 63% were from the county of Soest, 104 35% were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state Northrhine Westphalia and 6 2% were from five other federal states in Germany Figure 3 . Only eight 3% cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively Figure 4 . 75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 56% outbreak cases; 66 22% were confirmed by an increase in anti-phase II antibody titer CF , 89 30% had IgM antibodies against phase II antigens, 11 4% were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 44% cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two 11% of 18 cardiological patients and four 36% of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman case 3; Table 1 . Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23 . Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 . Touching the sheep was also significantly more common among cases 5/20 25% CCS2 cases vs. 0/22 0% controls; OR undefined; lower 95% CI = 1.1; p = 0.02 . 17 85% of 20 CCS2 cases, but only 6 25% of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was "having passed by the pen without stopping" OR = 17.0; 95%CI = 3.0-112.5; p < 0.01 . Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% . Fourty-five 56% were male and 35 44% were female. 13 16% met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen section 4, Figure 1 , 4 9% of 44 vendors became cases, compared to 2 13% of 15 persons who worked in section 1 p = 0.6 . Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 32% became cases. Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 . In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 25% tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market . More recently a large outbreak occurred in Jena Thuringia in 2005 with 322 reported cases associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature . An additional interview with the two cases with early onset 2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for "number of visitors", "proportion of adult visitors" and "attack rate among adults" see Table 3 . Displayed are attack rates and 95% confidence intervals. respectively could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep n = 1000-2000 the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic 50% 3/6 is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that in such a setting can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a "deprived" urban population "in touch" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author s declare that they have no competing interests.
What causes Q fever?	Coxiella burnetii (C. burnetii)	[ "BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii .", "To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii . RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away.", "The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters.", "The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3 rd trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii C. burnetii , a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle.", "C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats . High concentrations of C. burnetii can be found in birth products of infected mammals . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances .", "Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache.", "The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis . Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation.", "Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay IFA is considered to be the reference method, complement fixation CF , ELISA and microagglutination MA can also be used . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively .", "Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute.", "Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus.", "Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens ELISA or IFA , a 4-fold increase in anti-phase II IgG antibody titer ELISA or IFA or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors.", "Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case CCS1 case as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing.", "Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 AFE as OR-1 /OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context \"notified case\" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized.", "A case in this context \"notified case\" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition as used in the vendors' study s.b. identified through random sampling of the Soest population within CCS2 s.b. as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day.", "as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below \"Estimation of the overall attack rate\" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. .", "Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below \"Estimation of the overall attack rate\" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. . In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires.", "In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units.", "Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose 360° in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east Figure 1 . For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e.", "In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen in stand units or meters and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms.", "We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition as defined in the cohort study on vendors . The overall attack rate among adults was estimated based on the following sources: . Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later \"ill with fever\"; .", "Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later \"ill with fever\"; . Results of the small cohort of 9 sailor friends s.a. ; . Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever s.a. \"determination of outbreak size and descriptive epidemiology\" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source.", "Local health departments that identified outbreak cases of Q fever s.a. \"determination of outbreak size and descriptive epidemiology\" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in ., . and . we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors.", "we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children numerator . For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported.", "For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions number of visitors, proportion of adult visitors and clinical attack rate among adults we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks 70 animals . Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture.", "Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 CDC, Atlanta, USA . Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed.", "P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends.", "It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents.", "Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. Figure 2 . If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases mean +/-2 standard deviations had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female.", "The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female. Of the notified cases, 189 63% were from the county of Soest, 104 35% were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state Northrhine Westphalia and 6 2% were from five other federal states in Germany Figure 3 . Only eight 3% cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively Figure 4 . 75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized.", "75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 56% outbreak cases; 66 22% were confirmed by an increase in anti-phase II antibody titer CF , 89 30% had IgM antibodies against phase II antigens, 11 4% were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 44% cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever.", "18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two 11% of 18 cardiological patients and four 36% of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman case 3; Table 1 . Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections.", "Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23 . Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 .", "Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 . Touching the sheep was also significantly more common among cases 5/20 25% CCS2 cases vs. 0/22 0% controls; OR undefined; lower 95% CI = 1.1; p = 0.02 . 17 85% of 20 CCS2 cases, but only 6 25% of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was \"having passed by the pen without stopping\" OR = 17.0; 95%CI = 3.0-112.5; p < 0.01 . Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% .", "Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% . Fourty-five 56% were male and 35 44% were female. 13 16% met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2.", "Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen section 4, Figure 1 , 4 9% of 44 vendors became cases, compared to 2 13% of 15 persons who worked in section 1 p = 0.6 . Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 32% became cases. Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 .", "Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 . In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town.", "The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 25% tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise.", "The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market . More recently a large outbreak occurred in Jena Thuringia in 2005 with 322 reported cases associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately.", "The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature .", "The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature . An additional interview with the two cases with early onset 2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for \"number of visitors\", \"proportion of adult visitors\" and \"attack rate among adults\" see Table 3 . Displayed are attack rates and 95% confidence intervals.", "Scenarios 1-8 varied in the assumptions made for \"number of visitors\", \"proportion of adult visitors\" and \"attack rate among adults\" see Table 3 . Displayed are attack rates and 95% confidence intervals. respectively could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever.", "The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 .", "According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen.", "Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep.", "The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep n = 1000-2000 the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided .", "In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic 50% 3/6 is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system.", "In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults.", "Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks .", "Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that in such a setting can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a \"deprived\" urban population \"in touch\" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, .", "During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a \"deprived\" urban population \"in touch\" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals.", "Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author s declare that they have no competing interests." ]	1,583	5,207	BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii . RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3 rd trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii C. burnetii , a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats . High concentrations of C. burnetii can be found in birth products of infected mammals . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis . Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay IFA is considered to be the reference method, complement fixation CF , ELISA and microagglutination MA can also be used . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens ELISA or IFA , a 4-fold increase in anti-phase II IgG antibody titer ELISA or IFA or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case CCS1 case as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 AFE as OR-1 /OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context "notified case" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition as used in the vendors' study s.b. identified through random sampling of the Soest population within CCS2 s.b. as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below "Estimation of the overall attack rate" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. . In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose 360° in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east Figure 1 . For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen in stand units or meters and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition as defined in the cohort study on vendors . The overall attack rate among adults was estimated based on the following sources: . Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later "ill with fever"; . Results of the small cohort of 9 sailor friends s.a. ; . Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever s.a. "determination of outbreak size and descriptive epidemiology" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in ., . and . we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children numerator . For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions number of visitors, proportion of adult visitors and clinical attack rate among adults we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks 70 animals . Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 CDC, Atlanta, USA . Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. Figure 2 . If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases mean +/-2 standard deviations had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female. Of the notified cases, 189 63% were from the county of Soest, 104 35% were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state Northrhine Westphalia and 6 2% were from five other federal states in Germany Figure 3 . Only eight 3% cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively Figure 4 . 75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 56% outbreak cases; 66 22% were confirmed by an increase in anti-phase II antibody titer CF , 89 30% had IgM antibodies against phase II antigens, 11 4% were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 44% cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two 11% of 18 cardiological patients and four 36% of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman case 3; Table 1 . Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23 . Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 . Touching the sheep was also significantly more common among cases 5/20 25% CCS2 cases vs. 0/22 0% controls; OR undefined; lower 95% CI = 1.1; p = 0.02 . 17 85% of 20 CCS2 cases, but only 6 25% of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was "having passed by the pen without stopping" OR = 17.0; 95%CI = 3.0-112.5; p < 0.01 . Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% . Fourty-five 56% were male and 35 44% were female. 13 16% met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen section 4, Figure 1 , 4 9% of 44 vendors became cases, compared to 2 13% of 15 persons who worked in section 1 p = 0.6 . Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 32% became cases. Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 . In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 25% tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market . More recently a large outbreak occurred in Jena Thuringia in 2005 with 322 reported cases associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature . An additional interview with the two cases with early onset 2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for "number of visitors", "proportion of adult visitors" and "attack rate among adults" see Table 3 . Displayed are attack rates and 95% confidence intervals. respectively could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep n = 1000-2000 the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic 50% 3/6 is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that in such a setting can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a "deprived" urban population "in touch" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author s declare that they have no competing interests.
What is Coxiella burnetii?	small, gram-negative obligate intracellular bacterium	[ "BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii .", "To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii . RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away.", "The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters.", "The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3 rd trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii C. burnetii , a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle.", "C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats . High concentrations of C. burnetii can be found in birth products of infected mammals . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances .", "Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache.", "The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis . Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation.", "Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay IFA is considered to be the reference method, complement fixation CF , ELISA and microagglutination MA can also be used . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively .", "Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute.", "Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus.", "Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens ELISA or IFA , a 4-fold increase in anti-phase II IgG antibody titer ELISA or IFA or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors.", "Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case CCS1 case as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing.", "Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 AFE as OR-1 /OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context \"notified case\" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized.", "A case in this context \"notified case\" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition as used in the vendors' study s.b. identified through random sampling of the Soest population within CCS2 s.b. as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day.", "as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below \"Estimation of the overall attack rate\" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. .", "Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below \"Estimation of the overall attack rate\" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. . In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires.", "In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units.", "Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose 360° in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east Figure 1 . For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e.", "In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen in stand units or meters and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms.", "We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition as defined in the cohort study on vendors . The overall attack rate among adults was estimated based on the following sources: . Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later \"ill with fever\"; .", "Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later \"ill with fever\"; . Results of the small cohort of 9 sailor friends s.a. ; . Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever s.a. \"determination of outbreak size and descriptive epidemiology\" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source.", "Local health departments that identified outbreak cases of Q fever s.a. \"determination of outbreak size and descriptive epidemiology\" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in ., . and . we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors.", "we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children numerator . For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported.", "For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions number of visitors, proportion of adult visitors and clinical attack rate among adults we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks 70 animals . Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture.", "Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 CDC, Atlanta, USA . Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed.", "P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends.", "It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents.", "Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. Figure 2 . If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases mean +/-2 standard deviations had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female.", "The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female. Of the notified cases, 189 63% were from the county of Soest, 104 35% were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state Northrhine Westphalia and 6 2% were from five other federal states in Germany Figure 3 . Only eight 3% cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively Figure 4 . 75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized.", "75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 56% outbreak cases; 66 22% were confirmed by an increase in anti-phase II antibody titer CF , 89 30% had IgM antibodies against phase II antigens, 11 4% were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 44% cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever.", "18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two 11% of 18 cardiological patients and four 36% of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman case 3; Table 1 . Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections.", "Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23 . Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 .", "Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 . Touching the sheep was also significantly more common among cases 5/20 25% CCS2 cases vs. 0/22 0% controls; OR undefined; lower 95% CI = 1.1; p = 0.02 . 17 85% of 20 CCS2 cases, but only 6 25% of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was \"having passed by the pen without stopping\" OR = 17.0; 95%CI = 3.0-112.5; p < 0.01 . Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% .", "Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% . Fourty-five 56% were male and 35 44% were female. 13 16% met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2.", "Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen section 4, Figure 1 , 4 9% of 44 vendors became cases, compared to 2 13% of 15 persons who worked in section 1 p = 0.6 . Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 32% became cases. Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 .", "Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 . In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town.", "The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 25% tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise.", "The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market . More recently a large outbreak occurred in Jena Thuringia in 2005 with 322 reported cases associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately.", "The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature .", "The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature . An additional interview with the two cases with early onset 2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for \"number of visitors\", \"proportion of adult visitors\" and \"attack rate among adults\" see Table 3 . Displayed are attack rates and 95% confidence intervals.", "Scenarios 1-8 varied in the assumptions made for \"number of visitors\", \"proportion of adult visitors\" and \"attack rate among adults\" see Table 3 . Displayed are attack rates and 95% confidence intervals. respectively could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever.", "The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 .", "According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen.", "Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep.", "The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep n = 1000-2000 the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided .", "In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic 50% 3/6 is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system.", "In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults.", "Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks .", "Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that in such a setting can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a \"deprived\" urban population \"in touch\" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, .", "During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a \"deprived\" urban population \"in touch\" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals.", "Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author s declare that they have no competing interests." ]	1,583	5,208	BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii . RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3 rd trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii C. burnetii , a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats . High concentrations of C. burnetii can be found in birth products of infected mammals . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis . Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay IFA is considered to be the reference method, complement fixation CF , ELISA and microagglutination MA can also be used . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens ELISA or IFA , a 4-fold increase in anti-phase II IgG antibody titer ELISA or IFA or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case CCS1 case as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 AFE as OR-1 /OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context "notified case" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition as used in the vendors' study s.b. identified through random sampling of the Soest population within CCS2 s.b. as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below "Estimation of the overall attack rate" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. . In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose 360° in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east Figure 1 . For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen in stand units or meters and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition as defined in the cohort study on vendors . The overall attack rate among adults was estimated based on the following sources: . Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later "ill with fever"; . Results of the small cohort of 9 sailor friends s.a. ; . Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever s.a. "determination of outbreak size and descriptive epidemiology" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in ., . and . we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children numerator . For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions number of visitors, proportion of adult visitors and clinical attack rate among adults we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks 70 animals . Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 CDC, Atlanta, USA . Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. Figure 2 . If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases mean +/-2 standard deviations had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female. Of the notified cases, 189 63% were from the county of Soest, 104 35% were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state Northrhine Westphalia and 6 2% were from five other federal states in Germany Figure 3 . Only eight 3% cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively Figure 4 . 75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 56% outbreak cases; 66 22% were confirmed by an increase in anti-phase II antibody titer CF , 89 30% had IgM antibodies against phase II antigens, 11 4% were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 44% cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two 11% of 18 cardiological patients and four 36% of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman case 3; Table 1 . Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23 . Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 . Touching the sheep was also significantly more common among cases 5/20 25% CCS2 cases vs. 0/22 0% controls; OR undefined; lower 95% CI = 1.1; p = 0.02 . 17 85% of 20 CCS2 cases, but only 6 25% of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was "having passed by the pen without stopping" OR = 17.0; 95%CI = 3.0-112.5; p < 0.01 . Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% . Fourty-five 56% were male and 35 44% were female. 13 16% met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen section 4, Figure 1 , 4 9% of 44 vendors became cases, compared to 2 13% of 15 persons who worked in section 1 p = 0.6 . Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 32% became cases. Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 . In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 25% tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market . More recently a large outbreak occurred in Jena Thuringia in 2005 with 322 reported cases associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature . An additional interview with the two cases with early onset 2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for "number of visitors", "proportion of adult visitors" and "attack rate among adults" see Table 3 . Displayed are attack rates and 95% confidence intervals. respectively could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep n = 1000-2000 the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic 50% 3/6 is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that in such a setting can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a "deprived" urban population "in touch" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author s declare that they have no competing interests.
What is the primary reservoir for Coxiella burnetii?	sheep, goat and cattle	[ "BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii .", "To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii . RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away.", "The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters.", "The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3 rd trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii C. burnetii , a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle.", "C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats . High concentrations of C. burnetii can be found in birth products of infected mammals . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances .", "Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache.", "The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis . Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation.", "Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay IFA is considered to be the reference method, complement fixation CF , ELISA and microagglutination MA can also be used . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively .", "Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute.", "Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus.", "Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens ELISA or IFA , a 4-fold increase in anti-phase II IgG antibody titer ELISA or IFA or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors.", "Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case CCS1 case as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing.", "Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 AFE as OR-1 /OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context \"notified case\" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized.", "A case in this context \"notified case\" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition as used in the vendors' study s.b. identified through random sampling of the Soest population within CCS2 s.b. as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day.", "as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below \"Estimation of the overall attack rate\" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. .", "Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below \"Estimation of the overall attack rate\" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. . In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires.", "In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units.", "Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose 360° in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east Figure 1 . For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e.", "In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen in stand units or meters and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms.", "We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition as defined in the cohort study on vendors . The overall attack rate among adults was estimated based on the following sources: . Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later \"ill with fever\"; .", "Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later \"ill with fever\"; . Results of the small cohort of 9 sailor friends s.a. ; . Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever s.a. \"determination of outbreak size and descriptive epidemiology\" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source.", "Local health departments that identified outbreak cases of Q fever s.a. \"determination of outbreak size and descriptive epidemiology\" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in ., . and . we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors.", "we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children numerator . For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported.", "For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions number of visitors, proportion of adult visitors and clinical attack rate among adults we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks 70 animals . Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture.", "Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 CDC, Atlanta, USA . Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed.", "P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends.", "It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents.", "Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. Figure 2 . If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases mean +/-2 standard deviations had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female.", "The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female. Of the notified cases, 189 63% were from the county of Soest, 104 35% were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state Northrhine Westphalia and 6 2% were from five other federal states in Germany Figure 3 . Only eight 3% cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively Figure 4 . 75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized.", "75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 56% outbreak cases; 66 22% were confirmed by an increase in anti-phase II antibody titer CF , 89 30% had IgM antibodies against phase II antigens, 11 4% were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 44% cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever.", "18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two 11% of 18 cardiological patients and four 36% of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman case 3; Table 1 . Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections.", "Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23 . Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 .", "Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 . Touching the sheep was also significantly more common among cases 5/20 25% CCS2 cases vs. 0/22 0% controls; OR undefined; lower 95% CI = 1.1; p = 0.02 . 17 85% of 20 CCS2 cases, but only 6 25% of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was \"having passed by the pen without stopping\" OR = 17.0; 95%CI = 3.0-112.5; p < 0.01 . Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% .", "Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% . Fourty-five 56% were male and 35 44% were female. 13 16% met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2.", "Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen section 4, Figure 1 , 4 9% of 44 vendors became cases, compared to 2 13% of 15 persons who worked in section 1 p = 0.6 . Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 32% became cases. Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 .", "Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 . In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town.", "The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 25% tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise.", "The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market . More recently a large outbreak occurred in Jena Thuringia in 2005 with 322 reported cases associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately.", "The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature .", "The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature . An additional interview with the two cases with early onset 2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for \"number of visitors\", \"proportion of adult visitors\" and \"attack rate among adults\" see Table 3 . Displayed are attack rates and 95% confidence intervals.", "Scenarios 1-8 varied in the assumptions made for \"number of visitors\", \"proportion of adult visitors\" and \"attack rate among adults\" see Table 3 . Displayed are attack rates and 95% confidence intervals. respectively could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever.", "The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 .", "According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen.", "Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep.", "The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep n = 1000-2000 the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided .", "In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic 50% 3/6 is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system.", "In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults.", "Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks .", "Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that in such a setting can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a \"deprived\" urban population \"in touch\" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, .", "During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a \"deprived\" urban population \"in touch\" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals.", "Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author s declare that they have no competing interests." ]	1,583	5,209	BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii . RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3 rd trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii C. burnetii , a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats . High concentrations of C. burnetii can be found in birth products of infected mammals . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis . Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay IFA is considered to be the reference method, complement fixation CF , ELISA and microagglutination MA can also be used . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens ELISA or IFA , a 4-fold increase in anti-phase II IgG antibody titer ELISA or IFA or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case CCS1 case as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 AFE as OR-1 /OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context "notified case" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition as used in the vendors' study s.b. identified through random sampling of the Soest population within CCS2 s.b. as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below "Estimation of the overall attack rate" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. . In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose 360° in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east Figure 1 . For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen in stand units or meters and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition as defined in the cohort study on vendors . The overall attack rate among adults was estimated based on the following sources: . Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later "ill with fever"; . Results of the small cohort of 9 sailor friends s.a. ; . Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever s.a. "determination of outbreak size and descriptive epidemiology" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in ., . and . we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children numerator . For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions number of visitors, proportion of adult visitors and clinical attack rate among adults we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks 70 animals . Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 CDC, Atlanta, USA . Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. Figure 2 . If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases mean +/-2 standard deviations had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female. Of the notified cases, 189 63% were from the county of Soest, 104 35% were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state Northrhine Westphalia and 6 2% were from five other federal states in Germany Figure 3 . Only eight 3% cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively Figure 4 . 75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 56% outbreak cases; 66 22% were confirmed by an increase in anti-phase II antibody titer CF , 89 30% had IgM antibodies against phase II antigens, 11 4% were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 44% cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two 11% of 18 cardiological patients and four 36% of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman case 3; Table 1 . Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23 . Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 . Touching the sheep was also significantly more common among cases 5/20 25% CCS2 cases vs. 0/22 0% controls; OR undefined; lower 95% CI = 1.1; p = 0.02 . 17 85% of 20 CCS2 cases, but only 6 25% of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was "having passed by the pen without stopping" OR = 17.0; 95%CI = 3.0-112.5; p < 0.01 . Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% . Fourty-five 56% were male and 35 44% were female. 13 16% met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen section 4, Figure 1 , 4 9% of 44 vendors became cases, compared to 2 13% of 15 persons who worked in section 1 p = 0.6 . Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 32% became cases. Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 . In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 25% tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market . More recently a large outbreak occurred in Jena Thuringia in 2005 with 322 reported cases associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature . An additional interview with the two cases with early onset 2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for "number of visitors", "proportion of adult visitors" and "attack rate among adults" see Table 3 . Displayed are attack rates and 95% confidence intervals. respectively could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep n = 1000-2000 the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic 50% 3/6 is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that in such a setting can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a "deprived" urban population "in touch" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author s declare that they have no competing interests.
How are humans typically infected with Coxiella burnetii?	inhalation of contaminated aerosols from parturient fluids, placenta or wool	[ "BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii .", "To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii . RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away.", "The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters.", "The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3 rd trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii C. burnetii , a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle.", "C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats . High concentrations of C. burnetii can be found in birth products of infected mammals . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances .", "Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache.", "The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis . Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation.", "Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay IFA is considered to be the reference method, complement fixation CF , ELISA and microagglutination MA can also be used . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively .", "Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute.", "Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus.", "Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens ELISA or IFA , a 4-fold increase in anti-phase II IgG antibody titer ELISA or IFA or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors.", "Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case CCS1 case as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing.", "Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 AFE as OR-1 /OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context \"notified case\" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized.", "A case in this context \"notified case\" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition as used in the vendors' study s.b. identified through random sampling of the Soest population within CCS2 s.b. as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day.", "as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below \"Estimation of the overall attack rate\" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. .", "Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below \"Estimation of the overall attack rate\" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. . In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires.", "In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units.", "Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose 360° in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east Figure 1 . For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e.", "In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen in stand units or meters and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms.", "We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition as defined in the cohort study on vendors . The overall attack rate among adults was estimated based on the following sources: . Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later \"ill with fever\"; .", "Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later \"ill with fever\"; . Results of the small cohort of 9 sailor friends s.a. ; . Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever s.a. \"determination of outbreak size and descriptive epidemiology\" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source.", "Local health departments that identified outbreak cases of Q fever s.a. \"determination of outbreak size and descriptive epidemiology\" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in ., . and . we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors.", "we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children numerator . For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported.", "For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions number of visitors, proportion of adult visitors and clinical attack rate among adults we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks 70 animals . Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture.", "Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 CDC, Atlanta, USA . Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed.", "P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends.", "It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents.", "Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. Figure 2 . If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases mean +/-2 standard deviations had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female.", "The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female. Of the notified cases, 189 63% were from the county of Soest, 104 35% were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state Northrhine Westphalia and 6 2% were from five other federal states in Germany Figure 3 . Only eight 3% cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively Figure 4 . 75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized.", "75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 56% outbreak cases; 66 22% were confirmed by an increase in anti-phase II antibody titer CF , 89 30% had IgM antibodies against phase II antigens, 11 4% were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 44% cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever.", "18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two 11% of 18 cardiological patients and four 36% of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman case 3; Table 1 . Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections.", "Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23 . Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 .", "Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 . Touching the sheep was also significantly more common among cases 5/20 25% CCS2 cases vs. 0/22 0% controls; OR undefined; lower 95% CI = 1.1; p = 0.02 . 17 85% of 20 CCS2 cases, but only 6 25% of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was \"having passed by the pen without stopping\" OR = 17.0; 95%CI = 3.0-112.5; p < 0.01 . Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% .", "Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% . Fourty-five 56% were male and 35 44% were female. 13 16% met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2.", "Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen section 4, Figure 1 , 4 9% of 44 vendors became cases, compared to 2 13% of 15 persons who worked in section 1 p = 0.6 . Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 32% became cases. Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 .", "Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 . In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town.", "The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 25% tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise.", "The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market . More recently a large outbreak occurred in Jena Thuringia in 2005 with 322 reported cases associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately.", "The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature .", "The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature . An additional interview with the two cases with early onset 2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for \"number of visitors\", \"proportion of adult visitors\" and \"attack rate among adults\" see Table 3 . Displayed are attack rates and 95% confidence intervals.", "Scenarios 1-8 varied in the assumptions made for \"number of visitors\", \"proportion of adult visitors\" and \"attack rate among adults\" see Table 3 . Displayed are attack rates and 95% confidence intervals. respectively could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever.", "The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 .", "According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen.", "Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep.", "The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep n = 1000-2000 the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided .", "In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic 50% 3/6 is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system.", "In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults.", "Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks .", "Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that in such a setting can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a \"deprived\" urban population \"in touch\" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, .", "During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a \"deprived\" urban population \"in touch\" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals.", "Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author s declare that they have no competing interests." ]	1,583	5,210	BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study cases were Q fever patients, controls were randomly selected Soest citizens and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii C. burnetii . RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3 rd trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii C. burnetii , a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats . High concentrations of C. burnetii can be found in birth products of infected mammals . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool . Because the infectious dose is very low and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis . Children develop clinical disease less frequently . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay IFA is considered to be the reference method, complement fixation CF , ELISA and microagglutination MA can also be used . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year . In 2001 and 2002, 293 and 191 cases were notified, respectively . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory NCL for Coxiellae and the Robert Koch-Institute RKI , the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens ELISA or IFA , a 4-fold increase in anti-phase II IgG antibody titer ELISA or IFA or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case CCS1 case as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 AFE as OR-1 /OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context "notified case" was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition as used in the vendors' study s.b. identified through random sampling of the Soest population within CCS2 s.b. as well as in two cohorts vendors' study and the 9 sailor friends see below . The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market see below "Estimation of the overall attack rate" and gave an estimate of the proportion of clinically ill cases that were hospitalized s.a. . In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit approximately 3 meters . Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose 360° in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east Figure 1 . For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen plus minus 45° . Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen in stand units or meters and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition as defined in the cohort study on vendors . The overall attack rate among adults was estimated based on the following sources: . Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * . Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later "ill with fever"; . Results of the small cohort of 9 sailor friends s.a. ; . Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever s.a. "determination of outbreak size and descriptive epidemiology" interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in ., . and . we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children numerator . For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions number of visitors, proportion of adult visitors and clinical attack rate among adults we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks 70 animals . Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 CDC, Atlanta, USA . Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 65% of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. Figure 2 . If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases mean +/-2 standard deviations had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 53% were male and 140 47% were female. Of the notified cases, 189 63% were from the county of Soest, 104 35% were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state Northrhine Westphalia and 6 2% were from five other federal states in Germany Figure 3 . Only eight 3% cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively Figure 4 . 75 25% of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 21%; 95% CI = 6-46%; 1/5 CCS2 , 2/11 vendors study and 1/3 sailor friends clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 56% outbreak cases; 66 22% were confirmed by an increase in anti-phase II antibody titer CF , 89 30% had IgM antibodies against phase II antigens, 11 4% were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 44% cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two 11% of 18 cardiological patients and four 36% of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman case 3; Table 1 . Serological follow-up of the mothers detected chronic infection in the same woman case 3 12 weeks after delivery. One year follow-up of two newborn children of cases 1 and 3 identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23 . Seventeen 85% of 20 cases indicated that they had seen the cow that also was on display at the market next to the sheep compared to 7 32% of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 32% of 25 controls OR = 5.0; 95%CI = 1.2-22.3; p = 0.03 . Touching the sheep was also significantly more common among cases 5/20 25% CCS2 cases vs. 0/22 0% controls; OR undefined; lower 95% CI = 1.1; p = 0.02 . 17 85% of 20 CCS2 cases, but only 6 25% of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was "having passed by the pen without stopping" OR = 17.0; 95%CI = 3.0-112.5; p < 0.01 . Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 86% of 87 vendors, and received second hand information about 7 more overall response rate: 94% . Fourty-five 56% were male and 35 44% were female. 13 16% met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 55% became cases compared to only 7 10% of 70 persons who worked in a stand at a greater distance relative risk RR = 5.5 95%CI = 2.3-13.2; p = 0.002 ; Figure 1 . Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen section 4, Figure 1 , 4 9% of 44 vendors became cases, compared to 2 13% of 15 persons who worked in section 1 p = 0.6 . Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 32% became cases. Table 3 . In all scenarios the AR among adults was significantly higher than that among children Figure 5 . In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births 57 86% had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 25% tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% 8%; 12%; 12%; 16%; 24% . We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market . More recently a large outbreak occurred in Jena Thuringia in 2005 with 322 reported cases associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases source 5 were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature . An additional interview with the two cases with early onset 2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for "number of visitors", "proportion of adult visitors" and "attack rate among adults" see Table 3 . Displayed are attack rates and 95% confidence intervals. respectively could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to within 6 meters the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% 8/ 191 , 5% and 10% 4/39 , and there was at least one study with a much higher proportion 63% 10/ 16 . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep n = 1000-2000 the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep . In the most severely affected village, the clinical attack rate was 16% estimated from the data provided . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic 50% 3/6 is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks , was 40% compared to 1% in sheep flocks not linked to human outbreaks . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that in such a setting can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred , with more recent attempts to put a "deprived" urban population "in touch" with farm animals. Petting zoos, family farm vacations or the display of farm animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents 20, . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author s declare that they have no competing interests.
Who performed the sampling procedures?	veterinarians	[ "Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses.", "Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses.", "We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases.", "These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China.", "HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs .", "RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively.", "Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence.", "For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described .", "Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program .", "Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples .", "The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS.", "Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences.", "The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM.", "The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection.", "Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates.", "Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China.", "The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity .", "Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species.", "identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD .", "Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome.", "All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 .", "The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity .", "The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins.", "Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software.", "Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 .", "The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 .", "Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected.", "All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected.", "Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB.", "The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change .", "The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay.", "In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase.", "After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell.", "Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined.", "The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 .", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard .", "The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes.", "Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication.", "Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave.", "In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry.", "Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012.", "Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]	1,576	3,680	Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3
When were the fecal samples collected?	from November 2004 to November 2014	[ "Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses.", "Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses.", "We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases.", "These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China.", "HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs .", "RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively.", "Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence.", "For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described .", "Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program .", "Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples .", "The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS.", "Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences.", "The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM.", "The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection.", "Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates.", "Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China.", "The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity .", "Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species.", "identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD .", "Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome.", "All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 .", "The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity .", "The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins.", "Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software.", "Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 .", "The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 .", "Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected.", "All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected.", "Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB.", "The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change .", "The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay.", "In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase.", "After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell.", "Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined.", "The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 .", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard .", "The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes.", "Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication.", "Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave.", "In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry.", "Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012.", "Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]	1,576	3,681	Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3
What reference genome was used in the study?	BatCoV HKU10	[ "Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses.", "Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses.", "We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases.", "These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China.", "HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs .", "RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively.", "Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence.", "For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described .", "Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program .", "Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples .", "The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS.", "Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences.", "The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM.", "The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection.", "Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates.", "Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China.", "The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity .", "Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species.", "identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD .", "Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome.", "All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 .", "The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity .", "The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins.", "Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software.", "Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 .", "The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 .", "Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected.", "All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected.", "Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB.", "The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change .", "The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay.", "In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase.", "After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell.", "Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined.", "The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 .", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard .", "The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes.", "Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication.", "Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave.", "In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry.", "Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012.", "Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]	1,576	3,682	Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3
What is the length of the replicase gene ORF1ab?	20.4 kb	[ "Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses.", "Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses.", "We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases.", "These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China.", "HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs .", "RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively.", "Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence.", "For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described .", "Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program .", "Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples .", "The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS.", "Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences.", "The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM.", "The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection.", "Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates.", "Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China.", "The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity .", "Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species.", "identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD .", "Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome.", "All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 .", "The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity .", "The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins.", "Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software.", "Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 .", "The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 .", "Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected.", "All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected.", "Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB.", "The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change .", "The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay.", "In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase.", "After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell.", "Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined.", "The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 .", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard .", "The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes.", "Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication.", "Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave.", "In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry.", "Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012.", "Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]	1,576	3,684	Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3
What type of coronavirus was detected in R. affinis and R. sinicus species?	BtCoV/Rh/YN2012	[ "Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses.", "Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses.", "We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases.", "These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China.", "HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs .", "RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively.", "Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence.", "For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described .", "Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program .", "Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples .", "The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS.", "Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences.", "The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM.", "The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection.", "Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates.", "Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China.", "The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity .", "Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species.", "identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD .", "Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome.", "All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 .", "The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity .", "The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins.", "Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software.", "Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 .", "The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 .", "Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected.", "All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected.", "Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB.", "The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change .", "The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay.", "In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase.", "After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell.", "Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined.", "The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 .", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard .", "The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes.", "Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication.", "Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave.", "In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry.", "Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012.", "Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]	1,576	3,683	Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3
What is a natural reservoir of coronavirus?	Bats	[ "Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses.", "Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses.", "We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases.", "These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China.", "HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs .", "RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively.", "Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence.", "For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described .", "Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program .", "Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples .", "The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS.", "Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences.", "The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM.", "The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection.", "Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates.", "Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China.", "The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity .", "Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species.", "identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD .", "Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome.", "All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 .", "The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity .", "The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins.", "Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software.", "Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 .", "The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 .", "Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected.", "All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected.", "Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB.", "The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change .", "The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay.", "In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase.", "After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell.", "Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined.", "The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 .", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard .", "The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes.", "Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication.", "Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave.", "In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry.", "Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012.", "Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]	1,576	3,675	Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3
What is the genome size of the coronavirus?	26-32 kb	[ "Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses.", "Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses.", "We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases.", "These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China.", "HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs .", "RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively.", "Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence.", "For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described .", "Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program .", "Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples .", "The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS.", "Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences.", "The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM.", "The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection.", "Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates.", "Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China.", "The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity .", "Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species.", "identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD .", "Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome.", "All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 .", "The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity .", "The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins.", "Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software.", "Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 .", "The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 .", "Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected.", "All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected.", "Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB.", "The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change .", "The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay.", "In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase.", "After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell.", "Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined.", "The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 .", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard .", "The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes.", "Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication.", "Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave.", "In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry.", "Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012.", "Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]	1,576	3,676	Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3
What is the structure of the coronavirus?	enveloped, non-segmented, positive-strand RNA viruses	[ "Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses.", "Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses.", "We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases.", "These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China.", "HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs .", "RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively.", "Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence.", "For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described .", "Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program .", "Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples .", "The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS.", "Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences.", "The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM.", "The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection.", "Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates.", "Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China.", "The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity .", "Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species.", "identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD .", "Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome.", "All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 .", "The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity .", "The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins.", "Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software.", "Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 .", "The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 .", "Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected.", "All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected.", "Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB.", "The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change .", "The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay.", "In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase.", "After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell.", "Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined.", "The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 .", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard .", "The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes.", "Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication.", "Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave.", "In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry.", "Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012.", "Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]	1,576	3,677	Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3
What animals do gamma and delta coronavirus mainly infect?	birds	[ "Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses.", "Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses.", "We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases.", "These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China.", "HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs .", "RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively.", "Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence.", "For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described .", "Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program .", "Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples .", "The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS.", "Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences.", "The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM.", "The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection.", "Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates.", "Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China.", "The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity .", "Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species.", "identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD .", "Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome.", "All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 .", "The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity .", "The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins.", "Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software.", "Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 .", "The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 .", "Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected.", "All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected.", "Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB.", "The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change .", "The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay.", "In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase.", "After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell.", "Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined.", "The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 .", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard .", "The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes.", "Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication.", "Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave.", "In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry.", "Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012.", "Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]	1,576	3,678	Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3
How many types of coronaviruses are known to cause human disease?	Six	[ "Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses.", "Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses.", "We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases.", "These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China.", "HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs .", "RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively.", "Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence.", "For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described .", "Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program .", "Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples .", "The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS.", "Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences.", "The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM.", "The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection.", "Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates.", "Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China.", "The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity .", "Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species.", "identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD .", "Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome.", "All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 .", "The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity .", "The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins.", "Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software.", "Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 .", "The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 .", "Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected.", "All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected.", "Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB.", "The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change .", "The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay.", "In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase.", "After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell.", "Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined.", "The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 .", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard .", "The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes.", "Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication.", "Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave.", "In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry.", "Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012.", "Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]	1,576	3,679	Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3
What plays a role in regulating the immune response to a viral infection?	NF-κB	[ "Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses.", "Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses.", "We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases.", "These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China.", "HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs .", "RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively.", "Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence.", "For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described .", "Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program .", "Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples .", "The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS.", "Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences.", "The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM.", "The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection.", "Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates.", "Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China.", "The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity .", "Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species.", "identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD .", "Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome.", "All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 .", "The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity .", "The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins.", "Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software.", "Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 .", "The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 .", "Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected.", "All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected.", "Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB.", "The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change .", "The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay.", "In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase.", "After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell.", "Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined.", "The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 .", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard .", "The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes.", "Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication.", "Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave.", "In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry.", "Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012.", "Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]	1,576	3,685	Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3
What is the conclusion of the coronavirus long-term surveillance studies?	Rhinolophus bats seem to harbor a wide diversity of CoVs	[ "Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses.", "Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses.", "We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases.", "These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China.", "HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs .", "RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively.", "Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence.", "For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described .", "Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program .", "Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples .", "The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS.", "Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences.", "The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM.", "The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection.", "Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates.", "Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China.", "The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity .", "Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species.", "identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD .", "Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome.", "All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 .", "The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity .", "The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins.", "Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software.", "Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 .", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012.", "In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 .", "The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location.", "One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 .", "The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 .", "Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected.", "All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected.", "Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB.", "The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change .", "The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay.", "In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase.", "After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell.", "Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined.", "The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 .", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard .", "The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes.", "Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication.", "Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave.", "In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry.", "Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012.", "Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]	1,576	3,686	Bats have been identified as a natural reservoir of a variety of coronaviruses CoVs . Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV BtCoV/Rh/YN2012 in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae CoV , which consists of alpha, beta, gamma, and deltacoronaviruses CoVs . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals . Six human CoVs HCoVs are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus SARS-CoV and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology WIVH5210201 . The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit Roche Diagnostics GmbH, Mannheim, Germany as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription RT- PCR Invitrogen, San Diego, CA, USA was employed to detect coronavirus, as previously described . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b Cytob and/or NADH dehydrogenase subunit 1 ND1 gene using DNA extracted from the feces or swabs . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR Invitrogen, San Diego, CA, USA amplification with degenerate primers Table S1 designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase Invitrogen and Expand Long Template PCR System Roche Diagnostics GmbH, Mannheim, Germany with specific primers primer sequences are available upon request . Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA , respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector Promega, Madison, WI, USA for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing NGS data were filtered and mapped to the reference sequence of BatCoV HKU10 GenBank accession number NC_018871 using Geneious 7.1.8 . Genomes were preliminarily assembled using DNAStar lasergene V7 DNAStar, Madison, WI, USA . Putative open reading frames ORFs were predicted using NCBI's ORF finder orffinder/ with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region 5'-UTR and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence TRS were identified as previously described . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE . The aligned sequences were scanned for recombination events by using Recombination Detection Program . Potential recombination events as suggested by strong p-values <10 -20 were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform with default parameters . The protein homology detection was analyzed using HHpred with default parameters . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells Paki was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection ATCC, . The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously . The influenza virus NS1 plasmid was generated in our lab . The human bocavirus HBoV VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent Life Technologies . Expression of these accessory genes were analyzed by Western blotting using an mAb Roche Diagnostics GmbH, Mannheim, Germany against the HA tag. The virus isolation was performed as previously described . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic Gibco, Grand Island, NY, USA . Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR . Apoptosis was analyzed as previously described . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit YEASEN, Shanghai, China following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids pRL-TK and pIFN-βIFN-or pNF-κB-Luc , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 , SARS-CoV ORF7a , or HBoV VP2 . At 24 h post transfection, cells were treated with Sendai virus SeV 100 hemagglutinin units HAU /mL or human tumor necrosis factor alpha TNF-α; R&D system for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit Promega, Madison, WI, USA according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike mock were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species Figure 1A . CoVs were detected in 209 of these samples Figure 1B and Table 1 . Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 >94% nt identity , Mi-BatCoV HKU8 >93% nt identity , BtRf-AlphaCoV/HuB2013 >99% nt identity , SARSr-CoV >89% nt identity , and HKU2-related CoV >85% nt identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. identity . While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus Strain RsYN1, RsYN2, and RsYN3 , while the other one was from R.affinis Strain RaGD . The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences TRS that are identical to those of other alpha-CoVs Figure 2 and Table 2 . Exceptions including three additional ORFs ORF3b, ORF4a and ORF4b were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins Nsp1-Nsp16 . The 3'-end of the cleavage sites recognized by 3C-like proteinase Nsp4-Nsp10, Nsp12-Nsp16 and papain-like proteinase Nsp1-Nsp3 were confirmed. The proteins including Nsp3 papain-like 2 proteas, PL2pro , Nsp5 chymotrypsin-like protease, 3CLpro , Nsp12 RdRp , Nsp13 helicase , and other proteins of unknown function Table 3 . The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs Table 2 , suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes 83% genome identity correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan <75.4% genome identity . We then examined the individual genes Table 2 . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab >83.7% aa identity . Notably, the spike proteins are highly divergent among these strains. Other structure proteins E, M, and N are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes <65% aa identity . The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a 52.0-55.5% aa identity with BatCoV HKU10 ORF3 and ORF9 28.1-32.0% aa identity with SARSr-CoV ORF7a . We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues possibility: 100%, E value <10 −48 . We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing Figure 3 and Table 2 . The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs Figure 4 . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 0.727, 0.623, and 0.843, respectively were significantly higher than those of other ORFs Table 4 . For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a Figure S1 . ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 Figure S2 . The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids pIFNβ-Luc and pRL-TK and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 strain PR8 , HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 Figure 5A . Other accessory proteins showed no effect on IFN production Figure S3 . Expression of these accessory genes were confirmed by Western blot Figure S1 . Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. B ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector 500 ng , an NS1-expressing plasmid 500 ng , a SARS-CoV ORF7a-expressing plasmid 500 ng , or ORF3a-expressing plasmids 500 ng . After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate mean ± SD of fold change . Asterisks indicate significant differences between groups compared with Empty vector-NC, p < 0.05, as determined by student t test . NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids pNF-κB-Luc and pRL-TK , as well as accessory protein-expressing plasmids, or controls empty vector, NS1, SARS-CoV Tor2-ORF7a . The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 Figure 5B . Expressions of ORF3as were confirmed with Western blot Figure S1 . Other accessory proteins did not modulate NF-κB production Figure S4 . To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully Figure S5 . A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found Table S2 . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b only in RsYN1 , and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing , inhibition of cellular protein synthesis , cell-cycle blockage , and apoptosis induction . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3
What is the conclusion of the study?	the marmoset an appropriate animal model for biodefense-related pathogens	[ "The common marmoset Callithrix jacchus is increasingly being utilised as a nonhuman primate model for human disease, ranging from autoimmune to infectious disease. In order to fully exploit these models, meaningful comparison to the human host response is necessary. Commercially available reagents, primarily targeted to human cells, were utilised to assess the phenotype and activation status of key immune cell types and cytokines in naive and infected animals. Single cell suspensions of blood, spleen, and lung were examined. Generally, the phenotype of cells was comparable between humans and marmosets, with approximately 63% of all lymphocytes in the blood of marmosets being T cells, 25% B-cells, and 12% NK cells. The percentage of neutrophils in marmoset blood were more similar to human values than mouse values.", "Generally, the phenotype of cells was comparable between humans and marmosets, with approximately 63% of all lymphocytes in the blood of marmosets being T cells, 25% B-cells, and 12% NK cells. The percentage of neutrophils in marmoset blood were more similar to human values than mouse values. Comparison of the activation status of cells following experimental systemic or inhalational infection exhibited different trends in different tissues, most obvious in cell types active in the innate immune response. This work significantly enhances the ability to understand the immune response in these animals and fortifies their use as models of infectious disease. Text: The common marmoset Callithrix jacchus , a New World monkey NWM species is a small, arboreal nonhuman primate NHP , native to the Atlantic Coastal Forest in Northeast Brazil and parts of South East Brazil. In recent years the common marmoset has become more widely used in applied biomedical research, and an increasing body of evidence suggests the physiological and immunological responses to biological insults are similar between marmosets and humans .", "Text: The common marmoset Callithrix jacchus , a New World monkey NWM species is a small, arboreal nonhuman primate NHP , native to the Atlantic Coastal Forest in Northeast Brazil and parts of South East Brazil. In recent years the common marmoset has become more widely used in applied biomedical research, and an increasing body of evidence suggests the physiological and immunological responses to biological insults are similar between marmosets and humans . In the field of infectious disease, the marmoset is primarily being investigated as an alternative NHP model to complement the more traditionally used Old World monkeys OWM e.g., rhesus and cynomolgus macaques . Evolutionarily, both NWM and OWM sit within the simiiformes infraorder of the suborder Haplorhini of primates . Marmosets sit within the family Callitrichidae of the Platyrrhini parvorder, while OWM sit within the Cercopithecidae family of the Catarrhini Parvorder. Marmosets therefore are separated from Old World monkeys by one ancestral step and are a lower order primate.", "Marmosets sit within the family Callitrichidae of the Platyrrhini parvorder, while OWM sit within the Cercopithecidae family of the Catarrhini Parvorder. Marmosets therefore are separated from Old World monkeys by one ancestral step and are a lower order primate. Marmosets have been used to model the infection syndrome caused by a number of public health pathogens including Lassa virus , Hepatitis C virus , Dengue virus , Herpesvirus , Junin virus Rift Valley Fever , and SARS . Marmosets have also been used to model a number of biodefense pathogens including Eastern Equine Encephalitis virus , Bacillus anthracis , Francisella tularensis , Burkholderia pseudomallei , Marburg haemorrhagic fever virus , Ebola haemorrhagic fever virus , and Variola virus . The utility of marmosets to assess medical countermeasures has also been demonstrated; a vaccine has been tested for Lassa fever and the efficacy of ciprofloxacin and levofloxacin has been tested as postexposure therapies for anthrax and tularemia, respectively . In order to exploit these models fully and to allow meaningful comparison with the human condition, the response of the immune system to infection/therapy needs to be 2 Journal of Immunology Research characterised and understood.", "The utility of marmosets to assess medical countermeasures has also been demonstrated; a vaccine has been tested for Lassa fever and the efficacy of ciprofloxacin and levofloxacin has been tested as postexposure therapies for anthrax and tularemia, respectively . In order to exploit these models fully and to allow meaningful comparison with the human condition, the response of the immune system to infection/therapy needs to be 2 Journal of Immunology Research characterised and understood. Generally, NHPs have a close molecular, immunological, reproductive, and neurological similarity with humans making them ideal surrogates for humans and the study of infectious diseases. There is a high level of gene homology between humans and NHPs which underlies physiological and biochemical similarities. Similarities at the genetic level extend to the phenotypical level making NHPs well suited to modelling pathophysiological responses in man . Immunologically, there is a high degree of homology between humans and marmosets .", "Similarities at the genetic level extend to the phenotypical level making NHPs well suited to modelling pathophysiological responses in man . Immunologically, there is a high degree of homology between humans and marmosets . The similarity of various immunological factors produced by humans and marmosets has been investigated at both the genetic and protein levels. There is at least 95% homology between human costimulatory molecules e.g., CD80, CD86 etc. and those of marmosets . Also the immunoglobulin and T-cell receptor repertoire of humans and marmosets show at least 80% homology .", "and those of marmosets . Also the immunoglobulin and T-cell receptor repertoire of humans and marmosets show at least 80% homology . Currently, the availability of commercial reagents specifically designed for the marmoset is limited although a number of antibodies designed for use with human samples have been shown to cross-react with leucocytes from marmoset blood . However, these reagents have not been exploited to investigate the immune response to infectious disease. To date, investigation of the immune response in marmosets has primarily been achieved using pathogen-specific antibodies to determine the serological response using ELISA such as in the smallpox, Dengue, Rift Valley Fever, and Herpes models or by immunohistochemistry to identify, for example, CD8+, CD3+, CD20+ cells, and IL-6 in the smallpox model ; neutrophils and macrophages in the Herpes model ; or CD3+ and CD20+ cells in the Lassa model . The work presented here focuses on understanding the immune profile of the naive marmoset as well as identifying and quantifying the immune response to infectious disease.", "To date, investigation of the immune response in marmosets has primarily been achieved using pathogen-specific antibodies to determine the serological response using ELISA such as in the smallpox, Dengue, Rift Valley Fever, and Herpes models or by immunohistochemistry to identify, for example, CD8+, CD3+, CD20+ cells, and IL-6 in the smallpox model ; neutrophils and macrophages in the Herpes model ; or CD3+ and CD20+ cells in the Lassa model . The work presented here focuses on understanding the immune profile of the naive marmoset as well as identifying and quantifying the immune response to infectious disease. The aim of this work is to determine key changes and identify correlates of infection or protection. Healthy sexually mature common marmosets C. jacchus were obtained from the Dstl Porton Down breeding colony and housed in vasectomized male and female pairs. The Dstl colony was established during the 1970s and is a closed colony with a stable genotype. Animals included in these studies were mixed sex pairs, between 18 months and 5 years old and weighing between 320 g to 500 g. All animals were allowed free access to food and water as well as environmental enrichment.", "The Dstl colony was established during the 1970s and is a closed colony with a stable genotype. Animals included in these studies were mixed sex pairs, between 18 months and 5 years old and weighing between 320 g to 500 g. All animals were allowed free access to food and water as well as environmental enrichment. All animal studies were carried out in accordance with the UK Animals Scientific Procedures Act of 1986 and the Codes of Practice for the Housing and Care of Animals used in Scientific Procedures 1989. Animals were challenged with an intracellular pathogen by either the subcutaneous or inhalational route and were humanely killed at various time points after challenge. Prior to the infection study, animals were bled to determine baseline immunological parameters. Studies were performed to establish infection models in order to evaluate the efficacy of suitable therapies for transition ultimately to the clinic.", "Prior to the infection study, animals were bled to determine baseline immunological parameters. Studies were performed to establish infection models in order to evaluate the efficacy of suitable therapies for transition ultimately to the clinic. Populations. Blood and tissue samples were homogenised to provide single cell suspensions . Red blood cells were lysed, and the mixed leucocyte population was washed and stained with various combinations of the following fluorescent antibody stains: CD3 SP34-2 , CD8 LT8 , CD11c SHCL3 , CD14 M5E2 , CD16 3G8 , CD20 Bly1 , CD45RA 5H9 , CD54 HCD54 , CD56 B159 , CD69 FN50 , CD163 GHI/61 , and MCHII L243 BD Bioscience, Insight Bioscience, AbD serotec . Samples were fixed in 4% paraformaldehyde for 48 hrs at 4 ∘ C and analysed by flow cytometry FACScanto II BD within 72 hours of staining.", "Red blood cells were lysed, and the mixed leucocyte population was washed and stained with various combinations of the following fluorescent antibody stains: CD3 SP34-2 , CD8 LT8 , CD11c SHCL3 , CD14 M5E2 , CD16 3G8 , CD20 Bly1 , CD45RA 5H9 , CD54 HCD54 , CD56 B159 , CD69 FN50 , CD163 GHI/61 , and MCHII L243 BD Bioscience, Insight Bioscience, AbD serotec . Samples were fixed in 4% paraformaldehyde for 48 hrs at 4 ∘ C and analysed by flow cytometry FACScanto II BD within 72 hours of staining. Levels of circulating cytokines and chemokines were also quantified in the blood of marmosets from the Dstl colony using human multiplex kits available commercially BD cytokine flex beads and the Luminex system . These systems show significant cross-reactivity with the marmoset suggesting a high degree of conservation between the two species for IL-6, MIP-1 , MIP-1 , and MCP-1 . However, for other cytokines that are pivotal in the innate response, TNF and IFN reagents were obtained from U-CyTech Biosciences and Mabtech AB, respectively, due to a lack of cross-reactivity observed within the kit obtained from BD . In order to fully characterise the immune response to infectious agent in the marmoset, single cell suspensions of lung and spleen tissue were also examined in conjunction with the traditionally used blood cells.", "However, for other cytokines that are pivotal in the innate response, TNF and IFN reagents were obtained from U-CyTech Biosciences and Mabtech AB, respectively, due to a lack of cross-reactivity observed within the kit obtained from BD . In order to fully characterise the immune response to infectious agent in the marmoset, single cell suspensions of lung and spleen tissue were also examined in conjunction with the traditionally used blood cells. These tissue homogenates are of particular interest in relation to target sites of infection: the lung as the site of initial infection following an inhalational challenge and the spleen as a representative organ following a parental challenge. Cell types targeted during this analysis include cells important in the innate response e.g., neutrophils, macrophages, and NK cells and the adaptive response T and B cells with a view to determine the response to infection and vaccination and to derive immune correlates of infection/protection. Dapi was included as a nuclear marker to ensure that the initial gating included only intact cells. Basic cell types in blood were easily identified by measuring size forward and granularity side scatter Figure 1 a .", "Dapi was included as a nuclear marker to ensure that the initial gating included only intact cells. Basic cell types in blood were easily identified by measuring size forward and granularity side scatter Figure 1 a . Identification of cell types in tissue samples was more difficult as the scatter profiles are less clearly compartmentalized. The common leukocyte antigen CD45 normally used to locate all leukocytes in human samples also worked well in marmoset blood but failed to provide relevant information in the tissue samples. Confirmation of neutrophil identification was done by nuclear morphology and macrophages were identified by their adherent nature in initial experiments data not shown . Neutrophils were stained as CD11c dim CD14− and macrophages as CD11c + CD14+ regardless of tissue origin Figure 1 b .", "Confirmation of neutrophil identification was done by nuclear morphology and macrophages were identified by their adherent nature in initial experiments data not shown . Neutrophils were stained as CD11c dim CD14− and macrophages as CD11c + CD14+ regardless of tissue origin Figure 1 b . Figure 1 shows the basic division of lymphocytes between T, B, and NK cells from a healthy blood sample. Using this approach, the percentage of NK cells, B-cells, total T-cells, CD8+ T-cells, neutrophils, and monocytes was determined in the blood of naive marmosets Figure 2 a , Table 1 ; approximately 63% of all lymphocytes were T cells, 25% B cells, and 12% NK cells. The variability of the data is depicted in Figure 2 a with the greatest variability observed in the proportion of neutrophils. There were no obvious differences attributable to age or sex of the animals.", "The variability of the data is depicted in Figure 2 a with the greatest variability observed in the proportion of neutrophils. There were no obvious differences attributable to age or sex of the animals. This analysis was also applied to lung and spleen homogenates from naive marmosets Figures 2 b and 2 c . Greater variability was observed in the data relating to the identification of cell types in tissue samples, attributed to the inherent difficulties in identifying cell types in tissue homogenates by size and granularity and also the smaller cohort of animals. As expected, low numbers of neutrophils are found in naive spleen or lung tissue 8% both . Healthy mouse spleens typically have approximately 1-2% granulocytes .", "As expected, low numbers of neutrophils are found in naive spleen or lung tissue 8% both . Healthy mouse spleens typically have approximately 1-2% granulocytes . Understandably, there are few reports on the typical cell percentages expected in healthy human individuals for these tissues. However, it is reported that B cells are more prevalent in the spleens of humans at a ratio of 5 to 4 B to T cells than in the lungs which have a ratio of 1 to 8 B to T cells . In marmoset data reported here, a ratio of 2 to 3 B to T cells in the spleen and 1 to 6 B to T-cells in the lungs was observed compared to a ratio of 3 to 2 B to T cells in mouse spleens . Upon comparison, the marmoset data is generally consistent with previously reported data which is only available for marmoset blood samples and information available for human blood Table 1 .", "In marmoset data reported here, a ratio of 2 to 3 B to T cells in the spleen and 1 to 6 B to T-cells in the lungs was observed compared to a ratio of 3 to 2 B to T cells in mouse spleens . Upon comparison, the marmoset data is generally consistent with previously reported data which is only available for marmoset blood samples and information available for human blood Table 1 . However, one report found the proportion of CD8+ T-cells was almost three times greater in marmosets than humans, 61% to 21% respectively compared to the 30% observed in this study and the work previously reported by Brok et al. . Brok's study involved a small number of animals eight and also used a different CD8+ clone to identify cells. Contrastingly, in mice, differences are observed in the proportion of both B cells and neutrophils , although these differences are highly strain specific.", "Brok's study involved a small number of animals eight and also used a different CD8+ clone to identify cells. Contrastingly, in mice, differences are observed in the proportion of both B cells and neutrophils , although these differences are highly strain specific. C57BL/6J mice are reported to have 67% B cells and BALB/C mice 46%; both of which are consistently higher than the percentage found in marmosets and humans of approximately 25% Table 1 . The proportion of neutrophils found in the blood of C57BL/6J mice at 13% is lower than the 35% found in marmosets and the 40-75% expected for healthy human blood. This is encouraging as neutrophils play a pivotal role in the innate response to infection . A cross-species comparison suggests that monocytes comprise 3% of leukocytes Table 1 .", "This is encouraging as neutrophils play a pivotal role in the innate response to infection . A cross-species comparison suggests that monocytes comprise 3% of leukocytes Table 1 . Levels of circulating cytokines and chemokines IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN were also quantified in the blood, lung, and spleen of naïve marmosets from the Dstl colony. None of these cytokines were detected in blood samples from uninfected animals; however low levels of MIP-1 , MCP-1, and Rantes were found in spleen and lung tissue. Preliminary investigation of the immune response has supported the development of marmoset model of infection at Dstl. The levels of different cell types were measured at specific times after challenge with inhalational F. tularensis, B. pseudomallei, and Marburg virus .", "Preliminary investigation of the immune response has supported the development of marmoset model of infection at Dstl. The levels of different cell types were measured at specific times after challenge with inhalational F. tularensis, B. pseudomallei, and Marburg virus . Following challenge with F. tularensis, increasing levels of NK cells, neutrophils, T cells, and macrophages were observed, peaking at 48 hours after challenge before rapidly declining. This study also demonstrated the importance of investigating the immunological response in key target organs, as an increase in CD8+ T cells and T cells was observed in the spleen and lungs but not in the blood. Increasing levels of various cytokines, MCP-1, MIP-1 , MIP-1 , IL-6, and IL-1 , were observed in Table 1 : Comparison of the percentages of different cell types observed in the blood from healthy marmosets, mice, and humans. Identification markers Marmoset present data Marmoset Mouse 4 Human Asian Human Caucasian Number the lungs, spleen, and blood as the disease progressed TNF and IFN were not measured in this study .", "Increasing levels of various cytokines, MCP-1, MIP-1 , MIP-1 , IL-6, and IL-1 , were observed in Table 1 : Comparison of the percentages of different cell types observed in the blood from healthy marmosets, mice, and humans. Identification markers Marmoset present data Marmoset Mouse 4 Human Asian Human Caucasian Number the lungs, spleen, and blood as the disease progressed TNF and IFN were not measured in this study . Following inhalational challenge of marmosets with B. pseudomallei, an increase in the number of neutrophils was observed in the blood at 36 hours after challenge, followed by a rapid decline that was associated with an influx of neutrophils into the lung at 46 hours after challenge. A subsequent decline in the number of neutrophils in the lung was associated with the increased number in the spleen of animals that exhibited severe disease and were humanely killed. There was a gradual increase in the number of macrophages in the spleen as the disease progressed with numbers of macrophages peaking in the blood and lungs at 36 hours after challenge. A rapid decline in the number of macrophages in the lungs and blood was observed by 46 hours after challenge.", "There was a gradual increase in the number of macrophages in the spleen as the disease progressed with numbers of macrophages peaking in the blood and lungs at 36 hours after challenge. A rapid decline in the number of macrophages in the lungs and blood was observed by 46 hours after challenge. The levels of various cell types and cytokines were also measured in the blood of animals following inhalational challenge with Marburg virus . In these animals a general increase in the numbers of T cells, NK cells, macrophages IFN-, IL-1 , and MCP-1 was observed with time TNF was not measured . In order to gain more information from these acute bacterial infection models, we have sought out other markers from the literature. Primarily this was from marmoset models of autoimmune disorders such as rheumatoid arthritis and multiple sclerosis where the cross-reactivity of human antibodies was investigated, as well as the functionality of cells .", "In order to gain more information from these acute bacterial infection models, we have sought out other markers from the literature. Primarily this was from marmoset models of autoimmune disorders such as rheumatoid arthritis and multiple sclerosis where the cross-reactivity of human antibodies was investigated, as well as the functionality of cells . More recent work at Dstl has reported further cross-reactivity between marmoset cells and human cytokines to induce activity in marmoset T cells . These studies, combined with increasing information available on the cross-reactivity of human antibodies to various NHPs e.g., NIH NHP reagent resource, has expanded the ability to assess activation markers for disease. Detection of the following cell surface markers with human antibodies was trialed: CD54 ICAM-1 associated with cellular adhesion, inflammation, and leukocyte extravasation; CD69 the early activation marker; CD16 as a macrophage activation marker; CD163 the alternative macrophage activation marker; and MHC class II HLA-DR . CD56 was originally included to identify NK cells; however, it was noted that its expression on T cells was upregulated during disease and that cells defined as CD3+ CD16− CD56+ have been shown to be functionally cytotoxic in marmosets .", "Detection of the following cell surface markers with human antibodies was trialed: CD54 ICAM-1 associated with cellular adhesion, inflammation, and leukocyte extravasation; CD69 the early activation marker; CD16 as a macrophage activation marker; CD163 the alternative macrophage activation marker; and MHC class II HLA-DR . CD56 was originally included to identify NK cells; however, it was noted that its expression on T cells was upregulated during disease and that cells defined as CD3+ CD16− CD56+ have been shown to be functionally cytotoxic in marmosets . These markers have been used to expand on our previously published work to determine changes in the activation status of basic cell types in response to an acute bacterial infection. Animals were challenged with bacteria at a comparable dose either by inhalation = 22 or by a systemic route = 12 and humanely killed once they had reached a humane endpoint between day 4 and day 5 after challenge . Figure 3 illustrates the cellular activity in representative tissues following inhalational Figures 3 b and 3 e or systemic challenge Figures 3 c and 3 f and in naïve samples Figures 3 a and 3 d . Naïve T and NK cells appear to have similar resting activation states regardless of origin, whereas neutrophils and macrophages have differential expression of activation, for example, CD16.", "Figure 3 illustrates the cellular activity in representative tissues following inhalational Figures 3 b and 3 e or systemic challenge Figures 3 c and 3 f and in naïve samples Figures 3 a and 3 d . Naïve T and NK cells appear to have similar resting activation states regardless of origin, whereas neutrophils and macrophages have differential expression of activation, for example, CD16. In response to disease, the proportions of the cell types appear to remain relativity constant; however, the activation markers provide more detailed information and show involvement of all the cell types explored. Extensive activation was to be expected considering that the samples were taken at the humane endpoint. There is also extensive variation between The response to infection within the lungs has similarities across disease routes in terms of neutrophil reduced expression of CD16 and CD54 and macrophage increased expression of CD16 and reduction in MHCII. Unexpectedly, the T and NK cells appear to be more actively involved in systemic disease, indicating that the disease develops a pneumonic element regardless of initial route of infection.", "There is also extensive variation between The response to infection within the lungs has similarities across disease routes in terms of neutrophil reduced expression of CD16 and CD54 and macrophage increased expression of CD16 and reduction in MHCII. Unexpectedly, the T and NK cells appear to be more actively involved in systemic disease, indicating that the disease develops a pneumonic element regardless of initial route of infection. Levels of circulating cytokines and chemokines IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN were also quantified in the lung and spleen samples. All of the cytokines with the exception of Rantes were expressed at high levels ng/mg in all samples, which was expected as the animals had succumbed to terminal disease. The work presented here adds significant relevant information to the marmoset models of infection and to the understanding of the immune response in these animals. This work extends marmoset immunology from autoimmune disorders into the field of infectious diseases; this coupled with an increase in the information available on crossreactivity of human reagents to a variety of NHPs increases the utility/application of marmosets as models of human disease.", "The work presented here adds significant relevant information to the marmoset models of infection and to the understanding of the immune response in these animals. This work extends marmoset immunology from autoimmune disorders into the field of infectious diseases; this coupled with an increase in the information available on crossreactivity of human reagents to a variety of NHPs increases the utility/application of marmosets as models of human disease. In conclusion, the immune response in marmosets to infectious disease can be characterised in terms of the phenotype and activation status of all the major immune cells and key cytokine and chemokine expression. This can aid in the identification of correlates of infection or protection in medical countermeasures assessment studies. This information can also potentially be used for pivotal studies to support licensure of products under the FDA Animal Rule. This, in conjunction with the small size of marmosets, their immune response to infection that is comparable to humans, and the ability to house more statistically relevant numbers within high containment, makes the marmoset an appropriate animal model for biodefense-related pathogens." ]	1,587	5,230	The common marmoset Callithrix jacchus is increasingly being utilised as a nonhuman primate model for human disease, ranging from autoimmune to infectious disease. In order to fully exploit these models, meaningful comparison to the human host response is necessary. Commercially available reagents, primarily targeted to human cells, were utilised to assess the phenotype and activation status of key immune cell types and cytokines in naive and infected animals. Single cell suspensions of blood, spleen, and lung were examined. Generally, the phenotype of cells was comparable between humans and marmosets, with approximately 63% of all lymphocytes in the blood of marmosets being T cells, 25% B-cells, and 12% NK cells. The percentage of neutrophils in marmoset blood were more similar to human values than mouse values. Comparison of the activation status of cells following experimental systemic or inhalational infection exhibited different trends in different tissues, most obvious in cell types active in the innate immune response. This work significantly enhances the ability to understand the immune response in these animals and fortifies their use as models of infectious disease. Text: The common marmoset Callithrix jacchus , a New World monkey NWM species is a small, arboreal nonhuman primate NHP , native to the Atlantic Coastal Forest in Northeast Brazil and parts of South East Brazil. In recent years the common marmoset has become more widely used in applied biomedical research, and an increasing body of evidence suggests the physiological and immunological responses to biological insults are similar between marmosets and humans . In the field of infectious disease, the marmoset is primarily being investigated as an alternative NHP model to complement the more traditionally used Old World monkeys OWM e.g., rhesus and cynomolgus macaques . Evolutionarily, both NWM and OWM sit within the simiiformes infraorder of the suborder Haplorhini of primates . Marmosets sit within the family Callitrichidae of the Platyrrhini parvorder, while OWM sit within the Cercopithecidae family of the Catarrhini Parvorder. Marmosets therefore are separated from Old World monkeys by one ancestral step and are a lower order primate. Marmosets have been used to model the infection syndrome caused by a number of public health pathogens including Lassa virus , Hepatitis C virus , Dengue virus , Herpesvirus , Junin virus Rift Valley Fever , and SARS . Marmosets have also been used to model a number of biodefense pathogens including Eastern Equine Encephalitis virus , Bacillus anthracis , Francisella tularensis , Burkholderia pseudomallei , Marburg haemorrhagic fever virus , Ebola haemorrhagic fever virus , and Variola virus . The utility of marmosets to assess medical countermeasures has also been demonstrated; a vaccine has been tested for Lassa fever and the efficacy of ciprofloxacin and levofloxacin has been tested as postexposure therapies for anthrax and tularemia, respectively . In order to exploit these models fully and to allow meaningful comparison with the human condition, the response of the immune system to infection/therapy needs to be 2 Journal of Immunology Research characterised and understood. Generally, NHPs have a close molecular, immunological, reproductive, and neurological similarity with humans making them ideal surrogates for humans and the study of infectious diseases. There is a high level of gene homology between humans and NHPs which underlies physiological and biochemical similarities. Similarities at the genetic level extend to the phenotypical level making NHPs well suited to modelling pathophysiological responses in man . Immunologically, there is a high degree of homology between humans and marmosets . The similarity of various immunological factors produced by humans and marmosets has been investigated at both the genetic and protein levels. There is at least 95% homology between human costimulatory molecules e.g., CD80, CD86 etc. and those of marmosets . Also the immunoglobulin and T-cell receptor repertoire of humans and marmosets show at least 80% homology . Currently, the availability of commercial reagents specifically designed for the marmoset is limited although a number of antibodies designed for use with human samples have been shown to cross-react with leucocytes from marmoset blood . However, these reagents have not been exploited to investigate the immune response to infectious disease. To date, investigation of the immune response in marmosets has primarily been achieved using pathogen-specific antibodies to determine the serological response using ELISA such as in the smallpox, Dengue, Rift Valley Fever, and Herpes models or by immunohistochemistry to identify, for example, CD8+, CD3+, CD20+ cells, and IL-6 in the smallpox model ; neutrophils and macrophages in the Herpes model ; or CD3+ and CD20+ cells in the Lassa model . The work presented here focuses on understanding the immune profile of the naive marmoset as well as identifying and quantifying the immune response to infectious disease. The aim of this work is to determine key changes and identify correlates of infection or protection. Healthy sexually mature common marmosets C. jacchus were obtained from the Dstl Porton Down breeding colony and housed in vasectomized male and female pairs. The Dstl colony was established during the 1970s and is a closed colony with a stable genotype. Animals included in these studies were mixed sex pairs, between 18 months and 5 years old and weighing between 320 g to 500 g. All animals were allowed free access to food and water as well as environmental enrichment. All animal studies were carried out in accordance with the UK Animals Scientific Procedures Act of 1986 and the Codes of Practice for the Housing and Care of Animals used in Scientific Procedures 1989. Animals were challenged with an intracellular pathogen by either the subcutaneous or inhalational route and were humanely killed at various time points after challenge. Prior to the infection study, animals were bled to determine baseline immunological parameters. Studies were performed to establish infection models in order to evaluate the efficacy of suitable therapies for transition ultimately to the clinic. Populations. Blood and tissue samples were homogenised to provide single cell suspensions . Red blood cells were lysed, and the mixed leucocyte population was washed and stained with various combinations of the following fluorescent antibody stains: CD3 SP34-2 , CD8 LT8 , CD11c SHCL3 , CD14 M5E2 , CD16 3G8 , CD20 Bly1 , CD45RA 5H9 , CD54 HCD54 , CD56 B159 , CD69 FN50 , CD163 GHI/61 , and MCHII L243 BD Bioscience, Insight Bioscience, AbD serotec . Samples were fixed in 4% paraformaldehyde for 48 hrs at 4 ∘ C and analysed by flow cytometry FACScanto II BD within 72 hours of staining. Levels of circulating cytokines and chemokines were also quantified in the blood of marmosets from the Dstl colony using human multiplex kits available commercially BD cytokine flex beads and the Luminex system . These systems show significant cross-reactivity with the marmoset suggesting a high degree of conservation between the two species for IL-6, MIP-1 , MIP-1 , and MCP-1 . However, for other cytokines that are pivotal in the innate response, TNF and IFN reagents were obtained from U-CyTech Biosciences and Mabtech AB, respectively, due to a lack of cross-reactivity observed within the kit obtained from BD . In order to fully characterise the immune response to infectious agent in the marmoset, single cell suspensions of lung and spleen tissue were also examined in conjunction with the traditionally used blood cells. These tissue homogenates are of particular interest in relation to target sites of infection: the lung as the site of initial infection following an inhalational challenge and the spleen as a representative organ following a parental challenge. Cell types targeted during this analysis include cells important in the innate response e.g., neutrophils, macrophages, and NK cells and the adaptive response T and B cells with a view to determine the response to infection and vaccination and to derive immune correlates of infection/protection. Dapi was included as a nuclear marker to ensure that the initial gating included only intact cells. Basic cell types in blood were easily identified by measuring size forward and granularity side scatter Figure 1 a . Identification of cell types in tissue samples was more difficult as the scatter profiles are less clearly compartmentalized. The common leukocyte antigen CD45 normally used to locate all leukocytes in human samples also worked well in marmoset blood but failed to provide relevant information in the tissue samples. Confirmation of neutrophil identification was done by nuclear morphology and macrophages were identified by their adherent nature in initial experiments data not shown . Neutrophils were stained as CD11c dim CD14− and macrophages as CD11c + CD14+ regardless of tissue origin Figure 1 b . Figure 1 shows the basic division of lymphocytes between T, B, and NK cells from a healthy blood sample. Using this approach, the percentage of NK cells, B-cells, total T-cells, CD8+ T-cells, neutrophils, and monocytes was determined in the blood of naive marmosets Figure 2 a , Table 1 ; approximately 63% of all lymphocytes were T cells, 25% B cells, and 12% NK cells. The variability of the data is depicted in Figure 2 a with the greatest variability observed in the proportion of neutrophils. There were no obvious differences attributable to age or sex of the animals. This analysis was also applied to lung and spleen homogenates from naive marmosets Figures 2 b and 2 c . Greater variability was observed in the data relating to the identification of cell types in tissue samples, attributed to the inherent difficulties in identifying cell types in tissue homogenates by size and granularity and also the smaller cohort of animals. As expected, low numbers of neutrophils are found in naive spleen or lung tissue 8% both . Healthy mouse spleens typically have approximately 1-2% granulocytes . Understandably, there are few reports on the typical cell percentages expected in healthy human individuals for these tissues. However, it is reported that B cells are more prevalent in the spleens of humans at a ratio of 5 to 4 B to T cells than in the lungs which have a ratio of 1 to 8 B to T cells . In marmoset data reported here, a ratio of 2 to 3 B to T cells in the spleen and 1 to 6 B to T-cells in the lungs was observed compared to a ratio of 3 to 2 B to T cells in mouse spleens . Upon comparison, the marmoset data is generally consistent with previously reported data which is only available for marmoset blood samples and information available for human blood Table 1 . However, one report found the proportion of CD8+ T-cells was almost three times greater in marmosets than humans, 61% to 21% respectively compared to the 30% observed in this study and the work previously reported by Brok et al. . Brok's study involved a small number of animals eight and also used a different CD8+ clone to identify cells. Contrastingly, in mice, differences are observed in the proportion of both B cells and neutrophils , although these differences are highly strain specific. C57BL/6J mice are reported to have 67% B cells and BALB/C mice 46%; both of which are consistently higher than the percentage found in marmosets and humans of approximately 25% Table 1 . The proportion of neutrophils found in the blood of C57BL/6J mice at 13% is lower than the 35% found in marmosets and the 40-75% expected for healthy human blood. This is encouraging as neutrophils play a pivotal role in the innate response to infection . A cross-species comparison suggests that monocytes comprise 3% of leukocytes Table 1 . Levels of circulating cytokines and chemokines IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN were also quantified in the blood, lung, and spleen of naïve marmosets from the Dstl colony. None of these cytokines were detected in blood samples from uninfected animals; however low levels of MIP-1 , MCP-1, and Rantes were found in spleen and lung tissue. Preliminary investigation of the immune response has supported the development of marmoset model of infection at Dstl. The levels of different cell types were measured at specific times after challenge with inhalational F. tularensis, B. pseudomallei, and Marburg virus . Following challenge with F. tularensis, increasing levels of NK cells, neutrophils, T cells, and macrophages were observed, peaking at 48 hours after challenge before rapidly declining. This study also demonstrated the importance of investigating the immunological response in key target organs, as an increase in CD8+ T cells and T cells was observed in the spleen and lungs but not in the blood. Increasing levels of various cytokines, MCP-1, MIP-1 , MIP-1 , IL-6, and IL-1 , were observed in Table 1 : Comparison of the percentages of different cell types observed in the blood from healthy marmosets, mice, and humans. Identification markers Marmoset present data Marmoset Mouse 4 Human Asian Human Caucasian Number the lungs, spleen, and blood as the disease progressed TNF and IFN were not measured in this study . Following inhalational challenge of marmosets with B. pseudomallei, an increase in the number of neutrophils was observed in the blood at 36 hours after challenge, followed by a rapid decline that was associated with an influx of neutrophils into the lung at 46 hours after challenge. A subsequent decline in the number of neutrophils in the lung was associated with the increased number in the spleen of animals that exhibited severe disease and were humanely killed. There was a gradual increase in the number of macrophages in the spleen as the disease progressed with numbers of macrophages peaking in the blood and lungs at 36 hours after challenge. A rapid decline in the number of macrophages in the lungs and blood was observed by 46 hours after challenge. The levels of various cell types and cytokines were also measured in the blood of animals following inhalational challenge with Marburg virus . In these animals a general increase in the numbers of T cells, NK cells, macrophages IFN-, IL-1 , and MCP-1 was observed with time TNF was not measured . In order to gain more information from these acute bacterial infection models, we have sought out other markers from the literature. Primarily this was from marmoset models of autoimmune disorders such as rheumatoid arthritis and multiple sclerosis where the cross-reactivity of human antibodies was investigated, as well as the functionality of cells . More recent work at Dstl has reported further cross-reactivity between marmoset cells and human cytokines to induce activity in marmoset T cells . These studies, combined with increasing information available on the cross-reactivity of human antibodies to various NHPs e.g., NIH NHP reagent resource, has expanded the ability to assess activation markers for disease. Detection of the following cell surface markers with human antibodies was trialed: CD54 ICAM-1 associated with cellular adhesion, inflammation, and leukocyte extravasation; CD69 the early activation marker; CD16 as a macrophage activation marker; CD163 the alternative macrophage activation marker; and MHC class II HLA-DR . CD56 was originally included to identify NK cells; however, it was noted that its expression on T cells was upregulated during disease and that cells defined as CD3+ CD16− CD56+ have been shown to be functionally cytotoxic in marmosets . These markers have been used to expand on our previously published work to determine changes in the activation status of basic cell types in response to an acute bacterial infection. Animals were challenged with bacteria at a comparable dose either by inhalation = 22 or by a systemic route = 12 and humanely killed once they had reached a humane endpoint between day 4 and day 5 after challenge . Figure 3 illustrates the cellular activity in representative tissues following inhalational Figures 3 b and 3 e or systemic challenge Figures 3 c and 3 f and in naïve samples Figures 3 a and 3 d . Naïve T and NK cells appear to have similar resting activation states regardless of origin, whereas neutrophils and macrophages have differential expression of activation, for example, CD16. In response to disease, the proportions of the cell types appear to remain relativity constant; however, the activation markers provide more detailed information and show involvement of all the cell types explored. Extensive activation was to be expected considering that the samples were taken at the humane endpoint. There is also extensive variation between The response to infection within the lungs has similarities across disease routes in terms of neutrophil reduced expression of CD16 and CD54 and macrophage increased expression of CD16 and reduction in MHCII. Unexpectedly, the T and NK cells appear to be more actively involved in systemic disease, indicating that the disease develops a pneumonic element regardless of initial route of infection. Levels of circulating cytokines and chemokines IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN were also quantified in the lung and spleen samples. All of the cytokines with the exception of Rantes were expressed at high levels ng/mg in all samples, which was expected as the animals had succumbed to terminal disease. The work presented here adds significant relevant information to the marmoset models of infection and to the understanding of the immune response in these animals. This work extends marmoset immunology from autoimmune disorders into the field of infectious diseases; this coupled with an increase in the information available on crossreactivity of human reagents to a variety of NHPs increases the utility/application of marmosets as models of human disease. In conclusion, the immune response in marmosets to infectious disease can be characterised in terms of the phenotype and activation status of all the major immune cells and key cytokine and chemokine expression. This can aid in the identification of correlates of infection or protection in medical countermeasures assessment studies. This information can also potentially be used for pivotal studies to support licensure of products under the FDA Animal Rule. This, in conjunction with the small size of marmosets, their immune response to infection that is comparable to humans, and the ability to house more statistically relevant numbers within high containment, makes the marmoset an appropriate animal model for biodefense-related pathogens.
Why makes the marmoset an appropriate animal model for pathogen research?	the small size of marmosets, their immune response to infection that is comparable to humans, and the ability to house more statistically relevant numbers within high containment	[ "The common marmoset Callithrix jacchus is increasingly being utilised as a nonhuman primate model for human disease, ranging from autoimmune to infectious disease. In order to fully exploit these models, meaningful comparison to the human host response is necessary. Commercially available reagents, primarily targeted to human cells, were utilised to assess the phenotype and activation status of key immune cell types and cytokines in naive and infected animals. Single cell suspensions of blood, spleen, and lung were examined. Generally, the phenotype of cells was comparable between humans and marmosets, with approximately 63% of all lymphocytes in the blood of marmosets being T cells, 25% B-cells, and 12% NK cells. The percentage of neutrophils in marmoset blood were more similar to human values than mouse values.", "Generally, the phenotype of cells was comparable between humans and marmosets, with approximately 63% of all lymphocytes in the blood of marmosets being T cells, 25% B-cells, and 12% NK cells. The percentage of neutrophils in marmoset blood were more similar to human values than mouse values. Comparison of the activation status of cells following experimental systemic or inhalational infection exhibited different trends in different tissues, most obvious in cell types active in the innate immune response. This work significantly enhances the ability to understand the immune response in these animals and fortifies their use as models of infectious disease. Text: The common marmoset Callithrix jacchus , a New World monkey NWM species is a small, arboreal nonhuman primate NHP , native to the Atlantic Coastal Forest in Northeast Brazil and parts of South East Brazil. In recent years the common marmoset has become more widely used in applied biomedical research, and an increasing body of evidence suggests the physiological and immunological responses to biological insults are similar between marmosets and humans .", "Text: The common marmoset Callithrix jacchus , a New World monkey NWM species is a small, arboreal nonhuman primate NHP , native to the Atlantic Coastal Forest in Northeast Brazil and parts of South East Brazil. In recent years the common marmoset has become more widely used in applied biomedical research, and an increasing body of evidence suggests the physiological and immunological responses to biological insults are similar between marmosets and humans . In the field of infectious disease, the marmoset is primarily being investigated as an alternative NHP model to complement the more traditionally used Old World monkeys OWM e.g., rhesus and cynomolgus macaques . Evolutionarily, both NWM and OWM sit within the simiiformes infraorder of the suborder Haplorhini of primates . Marmosets sit within the family Callitrichidae of the Platyrrhini parvorder, while OWM sit within the Cercopithecidae family of the Catarrhini Parvorder. Marmosets therefore are separated from Old World monkeys by one ancestral step and are a lower order primate.", "Marmosets sit within the family Callitrichidae of the Platyrrhini parvorder, while OWM sit within the Cercopithecidae family of the Catarrhini Parvorder. Marmosets therefore are separated from Old World monkeys by one ancestral step and are a lower order primate. Marmosets have been used to model the infection syndrome caused by a number of public health pathogens including Lassa virus , Hepatitis C virus , Dengue virus , Herpesvirus , Junin virus Rift Valley Fever , and SARS . Marmosets have also been used to model a number of biodefense pathogens including Eastern Equine Encephalitis virus , Bacillus anthracis , Francisella tularensis , Burkholderia pseudomallei , Marburg haemorrhagic fever virus , Ebola haemorrhagic fever virus , and Variola virus . The utility of marmosets to assess medical countermeasures has also been demonstrated; a vaccine has been tested for Lassa fever and the efficacy of ciprofloxacin and levofloxacin has been tested as postexposure therapies for anthrax and tularemia, respectively . In order to exploit these models fully and to allow meaningful comparison with the human condition, the response of the immune system to infection/therapy needs to be 2 Journal of Immunology Research characterised and understood.", "The utility of marmosets to assess medical countermeasures has also been demonstrated; a vaccine has been tested for Lassa fever and the efficacy of ciprofloxacin and levofloxacin has been tested as postexposure therapies for anthrax and tularemia, respectively . In order to exploit these models fully and to allow meaningful comparison with the human condition, the response of the immune system to infection/therapy needs to be 2 Journal of Immunology Research characterised and understood. Generally, NHPs have a close molecular, immunological, reproductive, and neurological similarity with humans making them ideal surrogates for humans and the study of infectious diseases. There is a high level of gene homology between humans and NHPs which underlies physiological and biochemical similarities. Similarities at the genetic level extend to the phenotypical level making NHPs well suited to modelling pathophysiological responses in man . Immunologically, there is a high degree of homology between humans and marmosets .", "Similarities at the genetic level extend to the phenotypical level making NHPs well suited to modelling pathophysiological responses in man . Immunologically, there is a high degree of homology between humans and marmosets . The similarity of various immunological factors produced by humans and marmosets has been investigated at both the genetic and protein levels. There is at least 95% homology between human costimulatory molecules e.g., CD80, CD86 etc. and those of marmosets . Also the immunoglobulin and T-cell receptor repertoire of humans and marmosets show at least 80% homology .", "and those of marmosets . Also the immunoglobulin and T-cell receptor repertoire of humans and marmosets show at least 80% homology . Currently, the availability of commercial reagents specifically designed for the marmoset is limited although a number of antibodies designed for use with human samples have been shown to cross-react with leucocytes from marmoset blood . However, these reagents have not been exploited to investigate the immune response to infectious disease. To date, investigation of the immune response in marmosets has primarily been achieved using pathogen-specific antibodies to determine the serological response using ELISA such as in the smallpox, Dengue, Rift Valley Fever, and Herpes models or by immunohistochemistry to identify, for example, CD8+, CD3+, CD20+ cells, and IL-6 in the smallpox model ; neutrophils and macrophages in the Herpes model ; or CD3+ and CD20+ cells in the Lassa model . The work presented here focuses on understanding the immune profile of the naive marmoset as well as identifying and quantifying the immune response to infectious disease.", "To date, investigation of the immune response in marmosets has primarily been achieved using pathogen-specific antibodies to determine the serological response using ELISA such as in the smallpox, Dengue, Rift Valley Fever, and Herpes models or by immunohistochemistry to identify, for example, CD8+, CD3+, CD20+ cells, and IL-6 in the smallpox model ; neutrophils and macrophages in the Herpes model ; or CD3+ and CD20+ cells in the Lassa model . The work presented here focuses on understanding the immune profile of the naive marmoset as well as identifying and quantifying the immune response to infectious disease. The aim of this work is to determine key changes and identify correlates of infection or protection. Healthy sexually mature common marmosets C. jacchus were obtained from the Dstl Porton Down breeding colony and housed in vasectomized male and female pairs. The Dstl colony was established during the 1970s and is a closed colony with a stable genotype. Animals included in these studies were mixed sex pairs, between 18 months and 5 years old and weighing between 320 g to 500 g. All animals were allowed free access to food and water as well as environmental enrichment.", "The Dstl colony was established during the 1970s and is a closed colony with a stable genotype. Animals included in these studies were mixed sex pairs, between 18 months and 5 years old and weighing between 320 g to 500 g. All animals were allowed free access to food and water as well as environmental enrichment. All animal studies were carried out in accordance with the UK Animals Scientific Procedures Act of 1986 and the Codes of Practice for the Housing and Care of Animals used in Scientific Procedures 1989. Animals were challenged with an intracellular pathogen by either the subcutaneous or inhalational route and were humanely killed at various time points after challenge. Prior to the infection study, animals were bled to determine baseline immunological parameters. Studies were performed to establish infection models in order to evaluate the efficacy of suitable therapies for transition ultimately to the clinic.", "Prior to the infection study, animals were bled to determine baseline immunological parameters. Studies were performed to establish infection models in order to evaluate the efficacy of suitable therapies for transition ultimately to the clinic. Populations. Blood and tissue samples were homogenised to provide single cell suspensions . Red blood cells were lysed, and the mixed leucocyte population was washed and stained with various combinations of the following fluorescent antibody stains: CD3 SP34-2 , CD8 LT8 , CD11c SHCL3 , CD14 M5E2 , CD16 3G8 , CD20 Bly1 , CD45RA 5H9 , CD54 HCD54 , CD56 B159 , CD69 FN50 , CD163 GHI/61 , and MCHII L243 BD Bioscience, Insight Bioscience, AbD serotec . Samples were fixed in 4% paraformaldehyde for 48 hrs at 4 ∘ C and analysed by flow cytometry FACScanto II BD within 72 hours of staining.", "Red blood cells were lysed, and the mixed leucocyte population was washed and stained with various combinations of the following fluorescent antibody stains: CD3 SP34-2 , CD8 LT8 , CD11c SHCL3 , CD14 M5E2 , CD16 3G8 , CD20 Bly1 , CD45RA 5H9 , CD54 HCD54 , CD56 B159 , CD69 FN50 , CD163 GHI/61 , and MCHII L243 BD Bioscience, Insight Bioscience, AbD serotec . Samples were fixed in 4% paraformaldehyde for 48 hrs at 4 ∘ C and analysed by flow cytometry FACScanto II BD within 72 hours of staining. Levels of circulating cytokines and chemokines were also quantified in the blood of marmosets from the Dstl colony using human multiplex kits available commercially BD cytokine flex beads and the Luminex system . These systems show significant cross-reactivity with the marmoset suggesting a high degree of conservation between the two species for IL-6, MIP-1 , MIP-1 , and MCP-1 . However, for other cytokines that are pivotal in the innate response, TNF and IFN reagents were obtained from U-CyTech Biosciences and Mabtech AB, respectively, due to a lack of cross-reactivity observed within the kit obtained from BD . In order to fully characterise the immune response to infectious agent in the marmoset, single cell suspensions of lung and spleen tissue were also examined in conjunction with the traditionally used blood cells.", "However, for other cytokines that are pivotal in the innate response, TNF and IFN reagents were obtained from U-CyTech Biosciences and Mabtech AB, respectively, due to a lack of cross-reactivity observed within the kit obtained from BD . In order to fully characterise the immune response to infectious agent in the marmoset, single cell suspensions of lung and spleen tissue were also examined in conjunction with the traditionally used blood cells. These tissue homogenates are of particular interest in relation to target sites of infection: the lung as the site of initial infection following an inhalational challenge and the spleen as a representative organ following a parental challenge. Cell types targeted during this analysis include cells important in the innate response e.g., neutrophils, macrophages, and NK cells and the adaptive response T and B cells with a view to determine the response to infection and vaccination and to derive immune correlates of infection/protection. Dapi was included as a nuclear marker to ensure that the initial gating included only intact cells. Basic cell types in blood were easily identified by measuring size forward and granularity side scatter Figure 1 a .", "Dapi was included as a nuclear marker to ensure that the initial gating included only intact cells. Basic cell types in blood were easily identified by measuring size forward and granularity side scatter Figure 1 a . Identification of cell types in tissue samples was more difficult as the scatter profiles are less clearly compartmentalized. The common leukocyte antigen CD45 normally used to locate all leukocytes in human samples also worked well in marmoset blood but failed to provide relevant information in the tissue samples. Confirmation of neutrophil identification was done by nuclear morphology and macrophages were identified by their adherent nature in initial experiments data not shown . Neutrophils were stained as CD11c dim CD14− and macrophages as CD11c + CD14+ regardless of tissue origin Figure 1 b .", "Confirmation of neutrophil identification was done by nuclear morphology and macrophages were identified by their adherent nature in initial experiments data not shown . Neutrophils were stained as CD11c dim CD14− and macrophages as CD11c + CD14+ regardless of tissue origin Figure 1 b . Figure 1 shows the basic division of lymphocytes between T, B, and NK cells from a healthy blood sample. Using this approach, the percentage of NK cells, B-cells, total T-cells, CD8+ T-cells, neutrophils, and monocytes was determined in the blood of naive marmosets Figure 2 a , Table 1 ; approximately 63% of all lymphocytes were T cells, 25% B cells, and 12% NK cells. The variability of the data is depicted in Figure 2 a with the greatest variability observed in the proportion of neutrophils. There were no obvious differences attributable to age or sex of the animals.", "The variability of the data is depicted in Figure 2 a with the greatest variability observed in the proportion of neutrophils. There were no obvious differences attributable to age or sex of the animals. This analysis was also applied to lung and spleen homogenates from naive marmosets Figures 2 b and 2 c . Greater variability was observed in the data relating to the identification of cell types in tissue samples, attributed to the inherent difficulties in identifying cell types in tissue homogenates by size and granularity and also the smaller cohort of animals. As expected, low numbers of neutrophils are found in naive spleen or lung tissue 8% both . Healthy mouse spleens typically have approximately 1-2% granulocytes .", "As expected, low numbers of neutrophils are found in naive spleen or lung tissue 8% both . Healthy mouse spleens typically have approximately 1-2% granulocytes . Understandably, there are few reports on the typical cell percentages expected in healthy human individuals for these tissues. However, it is reported that B cells are more prevalent in the spleens of humans at a ratio of 5 to 4 B to T cells than in the lungs which have a ratio of 1 to 8 B to T cells . In marmoset data reported here, a ratio of 2 to 3 B to T cells in the spleen and 1 to 6 B to T-cells in the lungs was observed compared to a ratio of 3 to 2 B to T cells in mouse spleens . Upon comparison, the marmoset data is generally consistent with previously reported data which is only available for marmoset blood samples and information available for human blood Table 1 .", "In marmoset data reported here, a ratio of 2 to 3 B to T cells in the spleen and 1 to 6 B to T-cells in the lungs was observed compared to a ratio of 3 to 2 B to T cells in mouse spleens . Upon comparison, the marmoset data is generally consistent with previously reported data which is only available for marmoset blood samples and information available for human blood Table 1 . However, one report found the proportion of CD8+ T-cells was almost three times greater in marmosets than humans, 61% to 21% respectively compared to the 30% observed in this study and the work previously reported by Brok et al. . Brok's study involved a small number of animals eight and also used a different CD8+ clone to identify cells. Contrastingly, in mice, differences are observed in the proportion of both B cells and neutrophils , although these differences are highly strain specific.", "Brok's study involved a small number of animals eight and also used a different CD8+ clone to identify cells. Contrastingly, in mice, differences are observed in the proportion of both B cells and neutrophils , although these differences are highly strain specific. C57BL/6J mice are reported to have 67% B cells and BALB/C mice 46%; both of which are consistently higher than the percentage found in marmosets and humans of approximately 25% Table 1 . The proportion of neutrophils found in the blood of C57BL/6J mice at 13% is lower than the 35% found in marmosets and the 40-75% expected for healthy human blood. This is encouraging as neutrophils play a pivotal role in the innate response to infection . A cross-species comparison suggests that monocytes comprise 3% of leukocytes Table 1 .", "This is encouraging as neutrophils play a pivotal role in the innate response to infection . A cross-species comparison suggests that monocytes comprise 3% of leukocytes Table 1 . Levels of circulating cytokines and chemokines IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN were also quantified in the blood, lung, and spleen of naïve marmosets from the Dstl colony. None of these cytokines were detected in blood samples from uninfected animals; however low levels of MIP-1 , MCP-1, and Rantes were found in spleen and lung tissue. Preliminary investigation of the immune response has supported the development of marmoset model of infection at Dstl. The levels of different cell types were measured at specific times after challenge with inhalational F. tularensis, B. pseudomallei, and Marburg virus .", "Preliminary investigation of the immune response has supported the development of marmoset model of infection at Dstl. The levels of different cell types were measured at specific times after challenge with inhalational F. tularensis, B. pseudomallei, and Marburg virus . Following challenge with F. tularensis, increasing levels of NK cells, neutrophils, T cells, and macrophages were observed, peaking at 48 hours after challenge before rapidly declining. This study also demonstrated the importance of investigating the immunological response in key target organs, as an increase in CD8+ T cells and T cells was observed in the spleen and lungs but not in the blood. Increasing levels of various cytokines, MCP-1, MIP-1 , MIP-1 , IL-6, and IL-1 , were observed in Table 1 : Comparison of the percentages of different cell types observed in the blood from healthy marmosets, mice, and humans. Identification markers Marmoset present data Marmoset Mouse 4 Human Asian Human Caucasian Number the lungs, spleen, and blood as the disease progressed TNF and IFN were not measured in this study .", "Increasing levels of various cytokines, MCP-1, MIP-1 , MIP-1 , IL-6, and IL-1 , were observed in Table 1 : Comparison of the percentages of different cell types observed in the blood from healthy marmosets, mice, and humans. Identification markers Marmoset present data Marmoset Mouse 4 Human Asian Human Caucasian Number the lungs, spleen, and blood as the disease progressed TNF and IFN were not measured in this study . Following inhalational challenge of marmosets with B. pseudomallei, an increase in the number of neutrophils was observed in the blood at 36 hours after challenge, followed by a rapid decline that was associated with an influx of neutrophils into the lung at 46 hours after challenge. A subsequent decline in the number of neutrophils in the lung was associated with the increased number in the spleen of animals that exhibited severe disease and were humanely killed. There was a gradual increase in the number of macrophages in the spleen as the disease progressed with numbers of macrophages peaking in the blood and lungs at 36 hours after challenge. A rapid decline in the number of macrophages in the lungs and blood was observed by 46 hours after challenge.", "There was a gradual increase in the number of macrophages in the spleen as the disease progressed with numbers of macrophages peaking in the blood and lungs at 36 hours after challenge. A rapid decline in the number of macrophages in the lungs and blood was observed by 46 hours after challenge. The levels of various cell types and cytokines were also measured in the blood of animals following inhalational challenge with Marburg virus . In these animals a general increase in the numbers of T cells, NK cells, macrophages IFN-, IL-1 , and MCP-1 was observed with time TNF was not measured . In order to gain more information from these acute bacterial infection models, we have sought out other markers from the literature. Primarily this was from marmoset models of autoimmune disorders such as rheumatoid arthritis and multiple sclerosis where the cross-reactivity of human antibodies was investigated, as well as the functionality of cells .", "In order to gain more information from these acute bacterial infection models, we have sought out other markers from the literature. Primarily this was from marmoset models of autoimmune disorders such as rheumatoid arthritis and multiple sclerosis where the cross-reactivity of human antibodies was investigated, as well as the functionality of cells . More recent work at Dstl has reported further cross-reactivity between marmoset cells and human cytokines to induce activity in marmoset T cells . These studies, combined with increasing information available on the cross-reactivity of human antibodies to various NHPs e.g., NIH NHP reagent resource, has expanded the ability to assess activation markers for disease. Detection of the following cell surface markers with human antibodies was trialed: CD54 ICAM-1 associated with cellular adhesion, inflammation, and leukocyte extravasation; CD69 the early activation marker; CD16 as a macrophage activation marker; CD163 the alternative macrophage activation marker; and MHC class II HLA-DR . CD56 was originally included to identify NK cells; however, it was noted that its expression on T cells was upregulated during disease and that cells defined as CD3+ CD16− CD56+ have been shown to be functionally cytotoxic in marmosets .", "Detection of the following cell surface markers with human antibodies was trialed: CD54 ICAM-1 associated with cellular adhesion, inflammation, and leukocyte extravasation; CD69 the early activation marker; CD16 as a macrophage activation marker; CD163 the alternative macrophage activation marker; and MHC class II HLA-DR . CD56 was originally included to identify NK cells; however, it was noted that its expression on T cells was upregulated during disease and that cells defined as CD3+ CD16− CD56+ have been shown to be functionally cytotoxic in marmosets . These markers have been used to expand on our previously published work to determine changes in the activation status of basic cell types in response to an acute bacterial infection. Animals were challenged with bacteria at a comparable dose either by inhalation = 22 or by a systemic route = 12 and humanely killed once they had reached a humane endpoint between day 4 and day 5 after challenge . Figure 3 illustrates the cellular activity in representative tissues following inhalational Figures 3 b and 3 e or systemic challenge Figures 3 c and 3 f and in naïve samples Figures 3 a and 3 d . Naïve T and NK cells appear to have similar resting activation states regardless of origin, whereas neutrophils and macrophages have differential expression of activation, for example, CD16.", "Figure 3 illustrates the cellular activity in representative tissues following inhalational Figures 3 b and 3 e or systemic challenge Figures 3 c and 3 f and in naïve samples Figures 3 a and 3 d . Naïve T and NK cells appear to have similar resting activation states regardless of origin, whereas neutrophils and macrophages have differential expression of activation, for example, CD16. In response to disease, the proportions of the cell types appear to remain relativity constant; however, the activation markers provide more detailed information and show involvement of all the cell types explored. Extensive activation was to be expected considering that the samples were taken at the humane endpoint. There is also extensive variation between The response to infection within the lungs has similarities across disease routes in terms of neutrophil reduced expression of CD16 and CD54 and macrophage increased expression of CD16 and reduction in MHCII. Unexpectedly, the T and NK cells appear to be more actively involved in systemic disease, indicating that the disease develops a pneumonic element regardless of initial route of infection.", "There is also extensive variation between The response to infection within the lungs has similarities across disease routes in terms of neutrophil reduced expression of CD16 and CD54 and macrophage increased expression of CD16 and reduction in MHCII. Unexpectedly, the T and NK cells appear to be more actively involved in systemic disease, indicating that the disease develops a pneumonic element regardless of initial route of infection. Levels of circulating cytokines and chemokines IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN were also quantified in the lung and spleen samples. All of the cytokines with the exception of Rantes were expressed at high levels ng/mg in all samples, which was expected as the animals had succumbed to terminal disease. The work presented here adds significant relevant information to the marmoset models of infection and to the understanding of the immune response in these animals. This work extends marmoset immunology from autoimmune disorders into the field of infectious diseases; this coupled with an increase in the information available on crossreactivity of human reagents to a variety of NHPs increases the utility/application of marmosets as models of human disease.", "The work presented here adds significant relevant information to the marmoset models of infection and to the understanding of the immune response in these animals. This work extends marmoset immunology from autoimmune disorders into the field of infectious diseases; this coupled with an increase in the information available on crossreactivity of human reagents to a variety of NHPs increases the utility/application of marmosets as models of human disease. In conclusion, the immune response in marmosets to infectious disease can be characterised in terms of the phenotype and activation status of all the major immune cells and key cytokine and chemokine expression. This can aid in the identification of correlates of infection or protection in medical countermeasures assessment studies. This information can also potentially be used for pivotal studies to support licensure of products under the FDA Animal Rule. This, in conjunction with the small size of marmosets, their immune response to infection that is comparable to humans, and the ability to house more statistically relevant numbers within high containment, makes the marmoset an appropriate animal model for biodefense-related pathogens." ]	1,587	5,231	The common marmoset Callithrix jacchus is increasingly being utilised as a nonhuman primate model for human disease, ranging from autoimmune to infectious disease. In order to fully exploit these models, meaningful comparison to the human host response is necessary. Commercially available reagents, primarily targeted to human cells, were utilised to assess the phenotype and activation status of key immune cell types and cytokines in naive and infected animals. Single cell suspensions of blood, spleen, and lung were examined. Generally, the phenotype of cells was comparable between humans and marmosets, with approximately 63% of all lymphocytes in the blood of marmosets being T cells, 25% B-cells, and 12% NK cells. The percentage of neutrophils in marmoset blood were more similar to human values than mouse values. Comparison of the activation status of cells following experimental systemic or inhalational infection exhibited different trends in different tissues, most obvious in cell types active in the innate immune response. This work significantly enhances the ability to understand the immune response in these animals and fortifies their use as models of infectious disease. Text: The common marmoset Callithrix jacchus , a New World monkey NWM species is a small, arboreal nonhuman primate NHP , native to the Atlantic Coastal Forest in Northeast Brazil and parts of South East Brazil. In recent years the common marmoset has become more widely used in applied biomedical research, and an increasing body of evidence suggests the physiological and immunological responses to biological insults are similar between marmosets and humans . In the field of infectious disease, the marmoset is primarily being investigated as an alternative NHP model to complement the more traditionally used Old World monkeys OWM e.g., rhesus and cynomolgus macaques . Evolutionarily, both NWM and OWM sit within the simiiformes infraorder of the suborder Haplorhini of primates . Marmosets sit within the family Callitrichidae of the Platyrrhini parvorder, while OWM sit within the Cercopithecidae family of the Catarrhini Parvorder. Marmosets therefore are separated from Old World monkeys by one ancestral step and are a lower order primate. Marmosets have been used to model the infection syndrome caused by a number of public health pathogens including Lassa virus , Hepatitis C virus , Dengue virus , Herpesvirus , Junin virus Rift Valley Fever , and SARS . Marmosets have also been used to model a number of biodefense pathogens including Eastern Equine Encephalitis virus , Bacillus anthracis , Francisella tularensis , Burkholderia pseudomallei , Marburg haemorrhagic fever virus , Ebola haemorrhagic fever virus , and Variola virus . The utility of marmosets to assess medical countermeasures has also been demonstrated; a vaccine has been tested for Lassa fever and the efficacy of ciprofloxacin and levofloxacin has been tested as postexposure therapies for anthrax and tularemia, respectively . In order to exploit these models fully and to allow meaningful comparison with the human condition, the response of the immune system to infection/therapy needs to be 2 Journal of Immunology Research characterised and understood. Generally, NHPs have a close molecular, immunological, reproductive, and neurological similarity with humans making them ideal surrogates for humans and the study of infectious diseases. There is a high level of gene homology between humans and NHPs which underlies physiological and biochemical similarities. Similarities at the genetic level extend to the phenotypical level making NHPs well suited to modelling pathophysiological responses in man . Immunologically, there is a high degree of homology between humans and marmosets . The similarity of various immunological factors produced by humans and marmosets has been investigated at both the genetic and protein levels. There is at least 95% homology between human costimulatory molecules e.g., CD80, CD86 etc. and those of marmosets . Also the immunoglobulin and T-cell receptor repertoire of humans and marmosets show at least 80% homology . Currently, the availability of commercial reagents specifically designed for the marmoset is limited although a number of antibodies designed for use with human samples have been shown to cross-react with leucocytes from marmoset blood . However, these reagents have not been exploited to investigate the immune response to infectious disease. To date, investigation of the immune response in marmosets has primarily been achieved using pathogen-specific antibodies to determine the serological response using ELISA such as in the smallpox, Dengue, Rift Valley Fever, and Herpes models or by immunohistochemistry to identify, for example, CD8+, CD3+, CD20+ cells, and IL-6 in the smallpox model ; neutrophils and macrophages in the Herpes model ; or CD3+ and CD20+ cells in the Lassa model . The work presented here focuses on understanding the immune profile of the naive marmoset as well as identifying and quantifying the immune response to infectious disease. The aim of this work is to determine key changes and identify correlates of infection or protection. Healthy sexually mature common marmosets C. jacchus were obtained from the Dstl Porton Down breeding colony and housed in vasectomized male and female pairs. The Dstl colony was established during the 1970s and is a closed colony with a stable genotype. Animals included in these studies were mixed sex pairs, between 18 months and 5 years old and weighing between 320 g to 500 g. All animals were allowed free access to food and water as well as environmental enrichment. All animal studies were carried out in accordance with the UK Animals Scientific Procedures Act of 1986 and the Codes of Practice for the Housing and Care of Animals used in Scientific Procedures 1989. Animals were challenged with an intracellular pathogen by either the subcutaneous or inhalational route and were humanely killed at various time points after challenge. Prior to the infection study, animals were bled to determine baseline immunological parameters. Studies were performed to establish infection models in order to evaluate the efficacy of suitable therapies for transition ultimately to the clinic. Populations. Blood and tissue samples were homogenised to provide single cell suspensions . Red blood cells were lysed, and the mixed leucocyte population was washed and stained with various combinations of the following fluorescent antibody stains: CD3 SP34-2 , CD8 LT8 , CD11c SHCL3 , CD14 M5E2 , CD16 3G8 , CD20 Bly1 , CD45RA 5H9 , CD54 HCD54 , CD56 B159 , CD69 FN50 , CD163 GHI/61 , and MCHII L243 BD Bioscience, Insight Bioscience, AbD serotec . Samples were fixed in 4% paraformaldehyde for 48 hrs at 4 ∘ C and analysed by flow cytometry FACScanto II BD within 72 hours of staining. Levels of circulating cytokines and chemokines were also quantified in the blood of marmosets from the Dstl colony using human multiplex kits available commercially BD cytokine flex beads and the Luminex system . These systems show significant cross-reactivity with the marmoset suggesting a high degree of conservation between the two species for IL-6, MIP-1 , MIP-1 , and MCP-1 . However, for other cytokines that are pivotal in the innate response, TNF and IFN reagents were obtained from U-CyTech Biosciences and Mabtech AB, respectively, due to a lack of cross-reactivity observed within the kit obtained from BD . In order to fully characterise the immune response to infectious agent in the marmoset, single cell suspensions of lung and spleen tissue were also examined in conjunction with the traditionally used blood cells. These tissue homogenates are of particular interest in relation to target sites of infection: the lung as the site of initial infection following an inhalational challenge and the spleen as a representative organ following a parental challenge. Cell types targeted during this analysis include cells important in the innate response e.g., neutrophils, macrophages, and NK cells and the adaptive response T and B cells with a view to determine the response to infection and vaccination and to derive immune correlates of infection/protection. Dapi was included as a nuclear marker to ensure that the initial gating included only intact cells. Basic cell types in blood were easily identified by measuring size forward and granularity side scatter Figure 1 a . Identification of cell types in tissue samples was more difficult as the scatter profiles are less clearly compartmentalized. The common leukocyte antigen CD45 normally used to locate all leukocytes in human samples also worked well in marmoset blood but failed to provide relevant information in the tissue samples. Confirmation of neutrophil identification was done by nuclear morphology and macrophages were identified by their adherent nature in initial experiments data not shown . Neutrophils were stained as CD11c dim CD14− and macrophages as CD11c + CD14+ regardless of tissue origin Figure 1 b . Figure 1 shows the basic division of lymphocytes between T, B, and NK cells from a healthy blood sample. Using this approach, the percentage of NK cells, B-cells, total T-cells, CD8+ T-cells, neutrophils, and monocytes was determined in the blood of naive marmosets Figure 2 a , Table 1 ; approximately 63% of all lymphocytes were T cells, 25% B cells, and 12% NK cells. The variability of the data is depicted in Figure 2 a with the greatest variability observed in the proportion of neutrophils. There were no obvious differences attributable to age or sex of the animals. This analysis was also applied to lung and spleen homogenates from naive marmosets Figures 2 b and 2 c . Greater variability was observed in the data relating to the identification of cell types in tissue samples, attributed to the inherent difficulties in identifying cell types in tissue homogenates by size and granularity and also the smaller cohort of animals. As expected, low numbers of neutrophils are found in naive spleen or lung tissue 8% both . Healthy mouse spleens typically have approximately 1-2% granulocytes . Understandably, there are few reports on the typical cell percentages expected in healthy human individuals for these tissues. However, it is reported that B cells are more prevalent in the spleens of humans at a ratio of 5 to 4 B to T cells than in the lungs which have a ratio of 1 to 8 B to T cells . In marmoset data reported here, a ratio of 2 to 3 B to T cells in the spleen and 1 to 6 B to T-cells in the lungs was observed compared to a ratio of 3 to 2 B to T cells in mouse spleens . Upon comparison, the marmoset data is generally consistent with previously reported data which is only available for marmoset blood samples and information available for human blood Table 1 . However, one report found the proportion of CD8+ T-cells was almost three times greater in marmosets than humans, 61% to 21% respectively compared to the 30% observed in this study and the work previously reported by Brok et al. . Brok's study involved a small number of animals eight and also used a different CD8+ clone to identify cells. Contrastingly, in mice, differences are observed in the proportion of both B cells and neutrophils , although these differences are highly strain specific. C57BL/6J mice are reported to have 67% B cells and BALB/C mice 46%; both of which are consistently higher than the percentage found in marmosets and humans of approximately 25% Table 1 . The proportion of neutrophils found in the blood of C57BL/6J mice at 13% is lower than the 35% found in marmosets and the 40-75% expected for healthy human blood. This is encouraging as neutrophils play a pivotal role in the innate response to infection . A cross-species comparison suggests that monocytes comprise 3% of leukocytes Table 1 . Levels of circulating cytokines and chemokines IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN were also quantified in the blood, lung, and spleen of naïve marmosets from the Dstl colony. None of these cytokines were detected in blood samples from uninfected animals; however low levels of MIP-1 , MCP-1, and Rantes were found in spleen and lung tissue. Preliminary investigation of the immune response has supported the development of marmoset model of infection at Dstl. The levels of different cell types were measured at specific times after challenge with inhalational F. tularensis, B. pseudomallei, and Marburg virus . Following challenge with F. tularensis, increasing levels of NK cells, neutrophils, T cells, and macrophages were observed, peaking at 48 hours after challenge before rapidly declining. This study also demonstrated the importance of investigating the immunological response in key target organs, as an increase in CD8+ T cells and T cells was observed in the spleen and lungs but not in the blood. Increasing levels of various cytokines, MCP-1, MIP-1 , MIP-1 , IL-6, and IL-1 , were observed in Table 1 : Comparison of the percentages of different cell types observed in the blood from healthy marmosets, mice, and humans. Identification markers Marmoset present data Marmoset Mouse 4 Human Asian Human Caucasian Number the lungs, spleen, and blood as the disease progressed TNF and IFN were not measured in this study . Following inhalational challenge of marmosets with B. pseudomallei, an increase in the number of neutrophils was observed in the blood at 36 hours after challenge, followed by a rapid decline that was associated with an influx of neutrophils into the lung at 46 hours after challenge. A subsequent decline in the number of neutrophils in the lung was associated with the increased number in the spleen of animals that exhibited severe disease and were humanely killed. There was a gradual increase in the number of macrophages in the spleen as the disease progressed with numbers of macrophages peaking in the blood and lungs at 36 hours after challenge. A rapid decline in the number of macrophages in the lungs and blood was observed by 46 hours after challenge. The levels of various cell types and cytokines were also measured in the blood of animals following inhalational challenge with Marburg virus . In these animals a general increase in the numbers of T cells, NK cells, macrophages IFN-, IL-1 , and MCP-1 was observed with time TNF was not measured . In order to gain more information from these acute bacterial infection models, we have sought out other markers from the literature. Primarily this was from marmoset models of autoimmune disorders such as rheumatoid arthritis and multiple sclerosis where the cross-reactivity of human antibodies was investigated, as well as the functionality of cells . More recent work at Dstl has reported further cross-reactivity between marmoset cells and human cytokines to induce activity in marmoset T cells . These studies, combined with increasing information available on the cross-reactivity of human antibodies to various NHPs e.g., NIH NHP reagent resource, has expanded the ability to assess activation markers for disease. Detection of the following cell surface markers with human antibodies was trialed: CD54 ICAM-1 associated with cellular adhesion, inflammation, and leukocyte extravasation; CD69 the early activation marker; CD16 as a macrophage activation marker; CD163 the alternative macrophage activation marker; and MHC class II HLA-DR . CD56 was originally included to identify NK cells; however, it was noted that its expression on T cells was upregulated during disease and that cells defined as CD3+ CD16− CD56+ have been shown to be functionally cytotoxic in marmosets . These markers have been used to expand on our previously published work to determine changes in the activation status of basic cell types in response to an acute bacterial infection. Animals were challenged with bacteria at a comparable dose either by inhalation = 22 or by a systemic route = 12 and humanely killed once they had reached a humane endpoint between day 4 and day 5 after challenge . Figure 3 illustrates the cellular activity in representative tissues following inhalational Figures 3 b and 3 e or systemic challenge Figures 3 c and 3 f and in naïve samples Figures 3 a and 3 d . Naïve T and NK cells appear to have similar resting activation states regardless of origin, whereas neutrophils and macrophages have differential expression of activation, for example, CD16. In response to disease, the proportions of the cell types appear to remain relativity constant; however, the activation markers provide more detailed information and show involvement of all the cell types explored. Extensive activation was to be expected considering that the samples were taken at the humane endpoint. There is also extensive variation between The response to infection within the lungs has similarities across disease routes in terms of neutrophil reduced expression of CD16 and CD54 and macrophage increased expression of CD16 and reduction in MHCII. Unexpectedly, the T and NK cells appear to be more actively involved in systemic disease, indicating that the disease develops a pneumonic element regardless of initial route of infection. Levels of circulating cytokines and chemokines IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN were also quantified in the lung and spleen samples. All of the cytokines with the exception of Rantes were expressed at high levels ng/mg in all samples, which was expected as the animals had succumbed to terminal disease. The work presented here adds significant relevant information to the marmoset models of infection and to the understanding of the immune response in these animals. This work extends marmoset immunology from autoimmune disorders into the field of infectious diseases; this coupled with an increase in the information available on crossreactivity of human reagents to a variety of NHPs increases the utility/application of marmosets as models of human disease. In conclusion, the immune response in marmosets to infectious disease can be characterised in terms of the phenotype and activation status of all the major immune cells and key cytokine and chemokine expression. This can aid in the identification of correlates of infection or protection in medical countermeasures assessment studies. This information can also potentially be used for pivotal studies to support licensure of products under the FDA Animal Rule. This, in conjunction with the small size of marmosets, their immune response to infection that is comparable to humans, and the ability to house more statistically relevant numbers within high containment, makes the marmoset an appropriate animal model for biodefense-related pathogens.
What was the focus of this study?	the antiproliferative effect of a copper (II) complex on HT-29 colon cancer cells	[ "Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells.", "HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu II complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern .", "Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success .", "In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells .", "Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications .", "Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity .", "Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity . This study evaluated the anticancer potential of a copper II complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu BrHAP 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale.", "Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale. Cell viability was measured by a conventional MTT 3- 4,5-dimethylthiazol-2yl -2,5-diphenyltetrazolium bromide reduction assay. After 48 h exposure to six concentrations of Cu BrHAP 2 , cells were treated with MTT solution 2 mg/mL for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader Hidex, Turku, Finland . The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates.", "The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase LDH release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader.", "Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu BrHAP 2 was expressed as a percentage of the positive control. A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min.", "A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min. In brief, HT-29 cells 1 × 10 4 cells/well in 96-well plate were supplemented with Cu BrHAP 2 2 g/mL or DMSO negative control for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader Thermo Scientific . The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis.", "The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded.", "In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A 10 mg/mL and 50 L of propidium iodide PI 1 mg/mL were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer BD FACSCanto II . The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin .", "The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin . The plate was then supplemented with the working fluorescein solution 150 L , followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE .", "Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE . In brief, HT-29 cells 1 × 10 4 cells/mL were seeded in 96-well plates and treated with different concentrations of Cu BrHAP 2 and DMSO negative control for 24 h. After 30 min treatment with dihydroethidium DHE dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously .", "The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously . Plates with stained cells were analyzed using the ArrayScan HCS system Cellomics, PA, USA . Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit Promega, Madison, WI . HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme.", "HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. In brief, HT-29 cells 1.0 × 10 4 cells/well in a 96-well plate were treated with different concentrations of Cu BrHAP 2 for 3 h, followed by stimulation with TNF- 1 ng/mL for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate.", "After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends.", "All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends. An analysis of variance ANOVA was carried out using the prism statistical package GraphPad Software, USA . < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments.", "Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu BrHAP 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 .", "Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 . Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu BrHAP 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure.", "Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu BrHAP 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment Figure 3 . The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases.", "The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay.", "Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve AUC technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation.", "The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation. HT-29 cells were treated with different concentrations of Cu BrHAP 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation.", "As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu BrHAP 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells.", "Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway.", "To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor- TNF- . The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression .", "The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation . The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention.", "The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways . In the present study, the Cu BrHAP 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner.", "HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line CCD 841 showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu II compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation .", "Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry .", "Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis .", "This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis . As the main source of cellular ROS and adenosine triphosphate ATP , mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL.", "After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis.", "These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential MMP fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu BrHAP 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome .", "The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets .", "The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control.", "Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu BrHAP 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway." ]	1,607	5,277	Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu II complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity . This study evaluated the anticancer potential of a copper II complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu BrHAP 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale. Cell viability was measured by a conventional MTT 3- 4,5-dimethylthiazol-2yl -2,5-diphenyltetrazolium bromide reduction assay. After 48 h exposure to six concentrations of Cu BrHAP 2 , cells were treated with MTT solution 2 mg/mL for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader Hidex, Turku, Finland . The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase LDH release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu BrHAP 2 was expressed as a percentage of the positive control. A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min. In brief, HT-29 cells 1 × 10 4 cells/well in 96-well plate were supplemented with Cu BrHAP 2 2 g/mL or DMSO negative control for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader Thermo Scientific . The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A 10 mg/mL and 50 L of propidium iodide PI 1 mg/mL were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer BD FACSCanto II . The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin . The plate was then supplemented with the working fluorescein solution 150 L , followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE . In brief, HT-29 cells 1 × 10 4 cells/mL were seeded in 96-well plates and treated with different concentrations of Cu BrHAP 2 and DMSO negative control for 24 h. After 30 min treatment with dihydroethidium DHE dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously . Plates with stained cells were analyzed using the ArrayScan HCS system Cellomics, PA, USA . Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit Promega, Madison, WI . HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. In brief, HT-29 cells 1.0 × 10 4 cells/well in a 96-well plate were treated with different concentrations of Cu BrHAP 2 for 3 h, followed by stimulation with TNF- 1 ng/mL for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends. An analysis of variance ANOVA was carried out using the prism statistical package GraphPad Software, USA . < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu BrHAP 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 . Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu BrHAP 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu BrHAP 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment Figure 3 . The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve AUC technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation. HT-29 cells were treated with different concentrations of Cu BrHAP 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu BrHAP 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor- TNF- . The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation . The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways . In the present study, the Cu BrHAP 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line CCD 841 showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu II compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis . As the main source of cellular ROS and adenosine triphosphate ATP , mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential MMP fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu BrHAP 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu BrHAP 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway.
What is the third most prevalent cancer in females in the United States?	colorectal cancer	[ "Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells.", "HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu II complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern .", "Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success .", "In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells .", "Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications .", "Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity .", "Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity . This study evaluated the anticancer potential of a copper II complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu BrHAP 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale.", "Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale. Cell viability was measured by a conventional MTT 3- 4,5-dimethylthiazol-2yl -2,5-diphenyltetrazolium bromide reduction assay. After 48 h exposure to six concentrations of Cu BrHAP 2 , cells were treated with MTT solution 2 mg/mL for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader Hidex, Turku, Finland . The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates.", "The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase LDH release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader.", "Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu BrHAP 2 was expressed as a percentage of the positive control. A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min.", "A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min. In brief, HT-29 cells 1 × 10 4 cells/well in 96-well plate were supplemented with Cu BrHAP 2 2 g/mL or DMSO negative control for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader Thermo Scientific . The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis.", "The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded.", "In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A 10 mg/mL and 50 L of propidium iodide PI 1 mg/mL were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer BD FACSCanto II . The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin .", "The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin . The plate was then supplemented with the working fluorescein solution 150 L , followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE .", "Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE . In brief, HT-29 cells 1 × 10 4 cells/mL were seeded in 96-well plates and treated with different concentrations of Cu BrHAP 2 and DMSO negative control for 24 h. After 30 min treatment with dihydroethidium DHE dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously .", "The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously . Plates with stained cells were analyzed using the ArrayScan HCS system Cellomics, PA, USA . Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit Promega, Madison, WI . HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme.", "HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. In brief, HT-29 cells 1.0 × 10 4 cells/well in a 96-well plate were treated with different concentrations of Cu BrHAP 2 for 3 h, followed by stimulation with TNF- 1 ng/mL for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate.", "After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends.", "All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends. An analysis of variance ANOVA was carried out using the prism statistical package GraphPad Software, USA . < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments.", "Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu BrHAP 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 .", "Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 . Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu BrHAP 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure.", "Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu BrHAP 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment Figure 3 . The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases.", "The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay.", "Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve AUC technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation.", "The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation. HT-29 cells were treated with different concentrations of Cu BrHAP 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation.", "As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu BrHAP 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells.", "Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway.", "To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor- TNF- . The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression .", "The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation . The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention.", "The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways . In the present study, the Cu BrHAP 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner.", "HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line CCD 841 showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu II compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation .", "Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry .", "Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis .", "This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis . As the main source of cellular ROS and adenosine triphosphate ATP , mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL.", "After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis.", "These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential MMP fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu BrHAP 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome .", "The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets .", "The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control.", "Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu BrHAP 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway." ]	1,607	5,278	Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu II complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity . This study evaluated the anticancer potential of a copper II complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu BrHAP 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale. Cell viability was measured by a conventional MTT 3- 4,5-dimethylthiazol-2yl -2,5-diphenyltetrazolium bromide reduction assay. After 48 h exposure to six concentrations of Cu BrHAP 2 , cells were treated with MTT solution 2 mg/mL for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader Hidex, Turku, Finland . The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase LDH release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu BrHAP 2 was expressed as a percentage of the positive control. A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min. In brief, HT-29 cells 1 × 10 4 cells/well in 96-well plate were supplemented with Cu BrHAP 2 2 g/mL or DMSO negative control for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader Thermo Scientific . The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A 10 mg/mL and 50 L of propidium iodide PI 1 mg/mL were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer BD FACSCanto II . The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin . The plate was then supplemented with the working fluorescein solution 150 L , followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE . In brief, HT-29 cells 1 × 10 4 cells/mL were seeded in 96-well plates and treated with different concentrations of Cu BrHAP 2 and DMSO negative control for 24 h. After 30 min treatment with dihydroethidium DHE dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously . Plates with stained cells were analyzed using the ArrayScan HCS system Cellomics, PA, USA . Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit Promega, Madison, WI . HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. In brief, HT-29 cells 1.0 × 10 4 cells/well in a 96-well plate were treated with different concentrations of Cu BrHAP 2 for 3 h, followed by stimulation with TNF- 1 ng/mL for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends. An analysis of variance ANOVA was carried out using the prism statistical package GraphPad Software, USA . < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu BrHAP 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 . Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu BrHAP 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu BrHAP 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment Figure 3 . The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve AUC technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation. HT-29 cells were treated with different concentrations of Cu BrHAP 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu BrHAP 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor- TNF- . The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation . The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways . In the present study, the Cu BrHAP 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line CCD 841 showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu II compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis . As the main source of cellular ROS and adenosine triphosphate ATP , mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential MMP fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu BrHAP 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu BrHAP 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway.
What is the 1-year survival rate for colorectal cancer patients?	83.2%	[ "Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells.", "HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu II complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern .", "Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success .", "In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells .", "Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications .", "Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity .", "Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity . This study evaluated the anticancer potential of a copper II complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu BrHAP 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale.", "Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale. Cell viability was measured by a conventional MTT 3- 4,5-dimethylthiazol-2yl -2,5-diphenyltetrazolium bromide reduction assay. After 48 h exposure to six concentrations of Cu BrHAP 2 , cells were treated with MTT solution 2 mg/mL for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader Hidex, Turku, Finland . The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates.", "The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase LDH release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader.", "Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu BrHAP 2 was expressed as a percentage of the positive control. A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min.", "A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min. In brief, HT-29 cells 1 × 10 4 cells/well in 96-well plate were supplemented with Cu BrHAP 2 2 g/mL or DMSO negative control for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader Thermo Scientific . The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis.", "The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded.", "In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A 10 mg/mL and 50 L of propidium iodide PI 1 mg/mL were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer BD FACSCanto II . The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin .", "The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin . The plate was then supplemented with the working fluorescein solution 150 L , followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE .", "Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE . In brief, HT-29 cells 1 × 10 4 cells/mL were seeded in 96-well plates and treated with different concentrations of Cu BrHAP 2 and DMSO negative control for 24 h. After 30 min treatment with dihydroethidium DHE dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously .", "The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously . Plates with stained cells were analyzed using the ArrayScan HCS system Cellomics, PA, USA . Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit Promega, Madison, WI . HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme.", "HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. In brief, HT-29 cells 1.0 × 10 4 cells/well in a 96-well plate were treated with different concentrations of Cu BrHAP 2 for 3 h, followed by stimulation with TNF- 1 ng/mL for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate.", "After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends.", "All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends. An analysis of variance ANOVA was carried out using the prism statistical package GraphPad Software, USA . < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments.", "Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu BrHAP 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 .", "Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 . Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu BrHAP 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure.", "Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu BrHAP 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment Figure 3 . The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases.", "The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay.", "Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve AUC technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation.", "The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation. HT-29 cells were treated with different concentrations of Cu BrHAP 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation.", "As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu BrHAP 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells.", "Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway.", "To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor- TNF- . The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression .", "The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation . The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention.", "The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways . In the present study, the Cu BrHAP 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner.", "HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line CCD 841 showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu II compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation .", "Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry .", "Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis .", "This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis . As the main source of cellular ROS and adenosine triphosphate ATP , mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL.", "After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis.", "These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential MMP fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu BrHAP 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome .", "The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets .", "The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control.", "Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu BrHAP 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway." ]	1,607	5,279	Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu II complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity . This study evaluated the anticancer potential of a copper II complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu BrHAP 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale. Cell viability was measured by a conventional MTT 3- 4,5-dimethylthiazol-2yl -2,5-diphenyltetrazolium bromide reduction assay. After 48 h exposure to six concentrations of Cu BrHAP 2 , cells were treated with MTT solution 2 mg/mL for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader Hidex, Turku, Finland . The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase LDH release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu BrHAP 2 was expressed as a percentage of the positive control. A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min. In brief, HT-29 cells 1 × 10 4 cells/well in 96-well plate were supplemented with Cu BrHAP 2 2 g/mL or DMSO negative control for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader Thermo Scientific . The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A 10 mg/mL and 50 L of propidium iodide PI 1 mg/mL were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer BD FACSCanto II . The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin . The plate was then supplemented with the working fluorescein solution 150 L , followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE . In brief, HT-29 cells 1 × 10 4 cells/mL were seeded in 96-well plates and treated with different concentrations of Cu BrHAP 2 and DMSO negative control for 24 h. After 30 min treatment with dihydroethidium DHE dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously . Plates with stained cells were analyzed using the ArrayScan HCS system Cellomics, PA, USA . Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit Promega, Madison, WI . HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. In brief, HT-29 cells 1.0 × 10 4 cells/well in a 96-well plate were treated with different concentrations of Cu BrHAP 2 for 3 h, followed by stimulation with TNF- 1 ng/mL for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends. An analysis of variance ANOVA was carried out using the prism statistical package GraphPad Software, USA . < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu BrHAP 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 . Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu BrHAP 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu BrHAP 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment Figure 3 . The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve AUC technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation. HT-29 cells were treated with different concentrations of Cu BrHAP 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu BrHAP 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor- TNF- . The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation . The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways . In the present study, the Cu BrHAP 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line CCD 841 showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu II compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis . As the main source of cellular ROS and adenosine triphosphate ATP , mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential MMP fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu BrHAP 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu BrHAP 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway.
What is the 5-year survival rate for colorectal cancer patients?	64.3%	[ "Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells.", "HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu II complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern .", "Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success .", "In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells .", "Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications .", "Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity .", "Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity . This study evaluated the anticancer potential of a copper II complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu BrHAP 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale.", "Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale. Cell viability was measured by a conventional MTT 3- 4,5-dimethylthiazol-2yl -2,5-diphenyltetrazolium bromide reduction assay. After 48 h exposure to six concentrations of Cu BrHAP 2 , cells were treated with MTT solution 2 mg/mL for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader Hidex, Turku, Finland . The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates.", "The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase LDH release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader.", "Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu BrHAP 2 was expressed as a percentage of the positive control. A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min.", "A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min. In brief, HT-29 cells 1 × 10 4 cells/well in 96-well plate were supplemented with Cu BrHAP 2 2 g/mL or DMSO negative control for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader Thermo Scientific . The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis.", "The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded.", "In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A 10 mg/mL and 50 L of propidium iodide PI 1 mg/mL were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer BD FACSCanto II . The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin .", "The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin . The plate was then supplemented with the working fluorescein solution 150 L , followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE .", "Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE . In brief, HT-29 cells 1 × 10 4 cells/mL were seeded in 96-well plates and treated with different concentrations of Cu BrHAP 2 and DMSO negative control for 24 h. After 30 min treatment with dihydroethidium DHE dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously .", "The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously . Plates with stained cells were analyzed using the ArrayScan HCS system Cellomics, PA, USA . Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit Promega, Madison, WI . HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme.", "HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. In brief, HT-29 cells 1.0 × 10 4 cells/well in a 96-well plate were treated with different concentrations of Cu BrHAP 2 for 3 h, followed by stimulation with TNF- 1 ng/mL for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate.", "After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends.", "All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends. An analysis of variance ANOVA was carried out using the prism statistical package GraphPad Software, USA . < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments.", "Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu BrHAP 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 .", "Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 . Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu BrHAP 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure.", "Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu BrHAP 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment Figure 3 . The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases.", "The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay.", "Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve AUC technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation.", "The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation. HT-29 cells were treated with different concentrations of Cu BrHAP 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation.", "As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu BrHAP 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells.", "Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway.", "To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor- TNF- . The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression .", "The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation . The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention.", "The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways . In the present study, the Cu BrHAP 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner.", "HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line CCD 841 showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu II compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation .", "Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry .", "Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis .", "This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis . As the main source of cellular ROS and adenosine triphosphate ATP , mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL.", "After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis.", "These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential MMP fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu BrHAP 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome .", "The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets .", "The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control.", "Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu BrHAP 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway." ]	1,607	5,280	Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu II complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity . This study evaluated the anticancer potential of a copper II complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu BrHAP 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale. Cell viability was measured by a conventional MTT 3- 4,5-dimethylthiazol-2yl -2,5-diphenyltetrazolium bromide reduction assay. After 48 h exposure to six concentrations of Cu BrHAP 2 , cells were treated with MTT solution 2 mg/mL for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader Hidex, Turku, Finland . The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase LDH release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu BrHAP 2 was expressed as a percentage of the positive control. A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min. In brief, HT-29 cells 1 × 10 4 cells/well in 96-well plate were supplemented with Cu BrHAP 2 2 g/mL or DMSO negative control for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader Thermo Scientific . The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A 10 mg/mL and 50 L of propidium iodide PI 1 mg/mL were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer BD FACSCanto II . The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin . The plate was then supplemented with the working fluorescein solution 150 L , followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE . In brief, HT-29 cells 1 × 10 4 cells/mL were seeded in 96-well plates and treated with different concentrations of Cu BrHAP 2 and DMSO negative control for 24 h. After 30 min treatment with dihydroethidium DHE dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously . Plates with stained cells were analyzed using the ArrayScan HCS system Cellomics, PA, USA . Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit Promega, Madison, WI . HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. In brief, HT-29 cells 1.0 × 10 4 cells/well in a 96-well plate were treated with different concentrations of Cu BrHAP 2 for 3 h, followed by stimulation with TNF- 1 ng/mL for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends. An analysis of variance ANOVA was carried out using the prism statistical package GraphPad Software, USA . < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu BrHAP 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 . Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu BrHAP 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu BrHAP 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment Figure 3 . The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve AUC technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation. HT-29 cells were treated with different concentrations of Cu BrHAP 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu BrHAP 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor- TNF- . The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation . The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways . In the present study, the Cu BrHAP 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line CCD 841 showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu II compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis . As the main source of cellular ROS and adenosine triphosphate ATP , mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential MMP fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu BrHAP 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu BrHAP 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway.
How were nuclear morphological changes in HT-29 cells measured?	detection of nuclear condensation	[ "Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells.", "HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu II complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern .", "Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success .", "In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells .", "Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications .", "Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity .", "Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity . This study evaluated the anticancer potential of a copper II complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu BrHAP 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale.", "Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale. Cell viability was measured by a conventional MTT 3- 4,5-dimethylthiazol-2yl -2,5-diphenyltetrazolium bromide reduction assay. After 48 h exposure to six concentrations of Cu BrHAP 2 , cells were treated with MTT solution 2 mg/mL for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader Hidex, Turku, Finland . The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates.", "The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase LDH release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader.", "Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu BrHAP 2 was expressed as a percentage of the positive control. A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min.", "A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min. In brief, HT-29 cells 1 × 10 4 cells/well in 96-well plate were supplemented with Cu BrHAP 2 2 g/mL or DMSO negative control for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader Thermo Scientific . The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis.", "The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded.", "In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A 10 mg/mL and 50 L of propidium iodide PI 1 mg/mL were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer BD FACSCanto II . The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin .", "The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin . The plate was then supplemented with the working fluorescein solution 150 L , followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE .", "Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE . In brief, HT-29 cells 1 × 10 4 cells/mL were seeded in 96-well plates and treated with different concentrations of Cu BrHAP 2 and DMSO negative control for 24 h. After 30 min treatment with dihydroethidium DHE dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously .", "The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously . Plates with stained cells were analyzed using the ArrayScan HCS system Cellomics, PA, USA . Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit Promega, Madison, WI . HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme.", "HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. In brief, HT-29 cells 1.0 × 10 4 cells/well in a 96-well plate were treated with different concentrations of Cu BrHAP 2 for 3 h, followed by stimulation with TNF- 1 ng/mL for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate.", "After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends.", "All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends. An analysis of variance ANOVA was carried out using the prism statistical package GraphPad Software, USA . < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments.", "Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu BrHAP 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 .", "Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 . Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu BrHAP 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure.", "Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu BrHAP 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment Figure 3 . The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases.", "The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay.", "Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve AUC technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation.", "The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation. HT-29 cells were treated with different concentrations of Cu BrHAP 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation.", "As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu BrHAP 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells.", "Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway.", "To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor- TNF- . The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression .", "The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation . The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention.", "The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways . In the present study, the Cu BrHAP 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner.", "HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line CCD 841 showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu II compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation .", "Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry .", "Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis .", "This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis . As the main source of cellular ROS and adenosine triphosphate ATP , mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL.", "After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis.", "These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential MMP fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu BrHAP 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome .", "The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets .", "The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control.", "Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu BrHAP 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway." ]	1,607	5,281	Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu II complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity . This study evaluated the anticancer potential of a copper II complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu BrHAP 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale. Cell viability was measured by a conventional MTT 3- 4,5-dimethylthiazol-2yl -2,5-diphenyltetrazolium bromide reduction assay. After 48 h exposure to six concentrations of Cu BrHAP 2 , cells were treated with MTT solution 2 mg/mL for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader Hidex, Turku, Finland . The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase LDH release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu BrHAP 2 was expressed as a percentage of the positive control. A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min. In brief, HT-29 cells 1 × 10 4 cells/well in 96-well plate were supplemented with Cu BrHAP 2 2 g/mL or DMSO negative control for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader Thermo Scientific . The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A 10 mg/mL and 50 L of propidium iodide PI 1 mg/mL were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer BD FACSCanto II . The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin . The plate was then supplemented with the working fluorescein solution 150 L , followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE . In brief, HT-29 cells 1 × 10 4 cells/mL were seeded in 96-well plates and treated with different concentrations of Cu BrHAP 2 and DMSO negative control for 24 h. After 30 min treatment with dihydroethidium DHE dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously . Plates with stained cells were analyzed using the ArrayScan HCS system Cellomics, PA, USA . Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit Promega, Madison, WI . HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. In brief, HT-29 cells 1.0 × 10 4 cells/well in a 96-well plate were treated with different concentrations of Cu BrHAP 2 for 3 h, followed by stimulation with TNF- 1 ng/mL for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends. An analysis of variance ANOVA was carried out using the prism statistical package GraphPad Software, USA . < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu BrHAP 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 . Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu BrHAP 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu BrHAP 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment Figure 3 . The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve AUC technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation. HT-29 cells were treated with different concentrations of Cu BrHAP 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu BrHAP 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor- TNF- . The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation . The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways . In the present study, the Cu BrHAP 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line CCD 841 showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu II compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis . As the main source of cellular ROS and adenosine triphosphate ATP , mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential MMP fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu BrHAP 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu BrHAP 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway.
What is directly related to nuclear condensation?	apoptotic chromatin changes	[ "Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells.", "HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu II complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern .", "Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success .", "In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells .", "Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications .", "Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity .", "Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity . This study evaluated the anticancer potential of a copper II complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu BrHAP 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale.", "Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale. Cell viability was measured by a conventional MTT 3- 4,5-dimethylthiazol-2yl -2,5-diphenyltetrazolium bromide reduction assay. After 48 h exposure to six concentrations of Cu BrHAP 2 , cells were treated with MTT solution 2 mg/mL for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader Hidex, Turku, Finland . The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates.", "The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase LDH release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader.", "Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu BrHAP 2 was expressed as a percentage of the positive control. A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min.", "A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min. In brief, HT-29 cells 1 × 10 4 cells/well in 96-well plate were supplemented with Cu BrHAP 2 2 g/mL or DMSO negative control for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader Thermo Scientific . The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis.", "The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded.", "In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A 10 mg/mL and 50 L of propidium iodide PI 1 mg/mL were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer BD FACSCanto II . The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin .", "The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin . The plate was then supplemented with the working fluorescein solution 150 L , followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE .", "Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE . In brief, HT-29 cells 1 × 10 4 cells/mL were seeded in 96-well plates and treated with different concentrations of Cu BrHAP 2 and DMSO negative control for 24 h. After 30 min treatment with dihydroethidium DHE dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously .", "The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously . Plates with stained cells were analyzed using the ArrayScan HCS system Cellomics, PA, USA . Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit Promega, Madison, WI . HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme.", "HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. In brief, HT-29 cells 1.0 × 10 4 cells/well in a 96-well plate were treated with different concentrations of Cu BrHAP 2 for 3 h, followed by stimulation with TNF- 1 ng/mL for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate.", "After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends.", "All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends. An analysis of variance ANOVA was carried out using the prism statistical package GraphPad Software, USA . < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments.", "Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu BrHAP 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 .", "Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 . Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu BrHAP 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure.", "Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu BrHAP 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment Figure 3 . The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases.", "The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay.", "Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve AUC technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation.", "The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation. HT-29 cells were treated with different concentrations of Cu BrHAP 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation.", "As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu BrHAP 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells.", "Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway.", "To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor- TNF- . The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression .", "The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation . The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention.", "The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways . In the present study, the Cu BrHAP 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner.", "HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line CCD 841 showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu II compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation .", "Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry .", "Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis .", "This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis . As the main source of cellular ROS and adenosine triphosphate ATP , mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL.", "After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis.", "These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential MMP fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu BrHAP 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome .", "The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets .", "The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control.", "Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu BrHAP 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway." ]	1,607	5,282	Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu II complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity . This study evaluated the anticancer potential of a copper II complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu BrHAP 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale. Cell viability was measured by a conventional MTT 3- 4,5-dimethylthiazol-2yl -2,5-diphenyltetrazolium bromide reduction assay. After 48 h exposure to six concentrations of Cu BrHAP 2 , cells were treated with MTT solution 2 mg/mL for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader Hidex, Turku, Finland . The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase LDH release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu BrHAP 2 was expressed as a percentage of the positive control. A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min. In brief, HT-29 cells 1 × 10 4 cells/well in 96-well plate were supplemented with Cu BrHAP 2 2 g/mL or DMSO negative control for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader Thermo Scientific . The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A 10 mg/mL and 50 L of propidium iodide PI 1 mg/mL were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer BD FACSCanto II . The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin . The plate was then supplemented with the working fluorescein solution 150 L , followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE . In brief, HT-29 cells 1 × 10 4 cells/mL were seeded in 96-well plates and treated with different concentrations of Cu BrHAP 2 and DMSO negative control for 24 h. After 30 min treatment with dihydroethidium DHE dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously . Plates with stained cells were analyzed using the ArrayScan HCS system Cellomics, PA, USA . Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit Promega, Madison, WI . HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. In brief, HT-29 cells 1.0 × 10 4 cells/well in a 96-well plate were treated with different concentrations of Cu BrHAP 2 for 3 h, followed by stimulation with TNF- 1 ng/mL for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends. An analysis of variance ANOVA was carried out using the prism statistical package GraphPad Software, USA . < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu BrHAP 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 . Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu BrHAP 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu BrHAP 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment Figure 3 . The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve AUC technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation. HT-29 cells were treated with different concentrations of Cu BrHAP 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu BrHAP 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor- TNF- . The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation . The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways . In the present study, the Cu BrHAP 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line CCD 841 showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu II compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis . As the main source of cellular ROS and adenosine triphosphate ATP , mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential MMP fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu BrHAP 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu BrHAP 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway.
What morphological cell changes are most associated with apoptosis?	membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation	[ "Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells.", "HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu II complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern .", "Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success .", "In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells .", "Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications .", "Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity .", "Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity . This study evaluated the anticancer potential of a copper II complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu BrHAP 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale.", "Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale. Cell viability was measured by a conventional MTT 3- 4,5-dimethylthiazol-2yl -2,5-diphenyltetrazolium bromide reduction assay. After 48 h exposure to six concentrations of Cu BrHAP 2 , cells were treated with MTT solution 2 mg/mL for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader Hidex, Turku, Finland . The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates.", "The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase LDH release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader.", "Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu BrHAP 2 was expressed as a percentage of the positive control. A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min.", "A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min. In brief, HT-29 cells 1 × 10 4 cells/well in 96-well plate were supplemented with Cu BrHAP 2 2 g/mL or DMSO negative control for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader Thermo Scientific . The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis.", "The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded.", "In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A 10 mg/mL and 50 L of propidium iodide PI 1 mg/mL were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer BD FACSCanto II . The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin .", "The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin . The plate was then supplemented with the working fluorescein solution 150 L , followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE .", "Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE . In brief, HT-29 cells 1 × 10 4 cells/mL were seeded in 96-well plates and treated with different concentrations of Cu BrHAP 2 and DMSO negative control for 24 h. After 30 min treatment with dihydroethidium DHE dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously .", "The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously . Plates with stained cells were analyzed using the ArrayScan HCS system Cellomics, PA, USA . Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit Promega, Madison, WI . HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme.", "HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. In brief, HT-29 cells 1.0 × 10 4 cells/well in a 96-well plate were treated with different concentrations of Cu BrHAP 2 for 3 h, followed by stimulation with TNF- 1 ng/mL for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate.", "After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends.", "All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends. An analysis of variance ANOVA was carried out using the prism statistical package GraphPad Software, USA . < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments.", "Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu BrHAP 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 .", "Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 . Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu BrHAP 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure.", "Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu BrHAP 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment Figure 3 . The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases.", "The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay.", "Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve AUC technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation.", "The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation. HT-29 cells were treated with different concentrations of Cu BrHAP 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation.", "As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu BrHAP 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells.", "Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway.", "To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor- TNF- . The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression .", "The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation . The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention.", "The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways . In the present study, the Cu BrHAP 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner.", "HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line CCD 841 showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu II compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation .", "Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry .", "Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis .", "This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis . As the main source of cellular ROS and adenosine triphosphate ATP , mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL.", "After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis.", "These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential MMP fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu BrHAP 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome .", "The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets .", "The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control.", "Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu BrHAP 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway." ]	1,607	5,283	Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu II complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity . This study evaluated the anticancer potential of a copper II complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu BrHAP 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale. Cell viability was measured by a conventional MTT 3- 4,5-dimethylthiazol-2yl -2,5-diphenyltetrazolium bromide reduction assay. After 48 h exposure to six concentrations of Cu BrHAP 2 , cells were treated with MTT solution 2 mg/mL for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader Hidex, Turku, Finland . The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase LDH release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu BrHAP 2 was expressed as a percentage of the positive control. A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min. In brief, HT-29 cells 1 × 10 4 cells/well in 96-well plate were supplemented with Cu BrHAP 2 2 g/mL or DMSO negative control for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader Thermo Scientific . The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A 10 mg/mL and 50 L of propidium iodide PI 1 mg/mL were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer BD FACSCanto II . The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin . The plate was then supplemented with the working fluorescein solution 150 L , followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE . In brief, HT-29 cells 1 × 10 4 cells/mL were seeded in 96-well plates and treated with different concentrations of Cu BrHAP 2 and DMSO negative control for 24 h. After 30 min treatment with dihydroethidium DHE dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously . Plates with stained cells were analyzed using the ArrayScan HCS system Cellomics, PA, USA . Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit Promega, Madison, WI . HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. In brief, HT-29 cells 1.0 × 10 4 cells/well in a 96-well plate were treated with different concentrations of Cu BrHAP 2 for 3 h, followed by stimulation with TNF- 1 ng/mL for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends. An analysis of variance ANOVA was carried out using the prism statistical package GraphPad Software, USA . < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu BrHAP 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 . Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu BrHAP 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu BrHAP 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment Figure 3 . The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve AUC technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation. HT-29 cells were treated with different concentrations of Cu BrHAP 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu BrHAP 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor- TNF- . The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation . The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways . In the present study, the Cu BrHAP 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line CCD 841 showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu II compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis . As the main source of cellular ROS and adenosine triphosphate ATP , mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential MMP fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu BrHAP 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu BrHAP 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway.
What types of cells are suitable for colon cancer studies?	HT-29 cells	[ "Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells.", "HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu II complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern .", "Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success .", "In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells .", "Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications .", "Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity .", "Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity . This study evaluated the anticancer potential of a copper II complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu BrHAP 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale.", "Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale. Cell viability was measured by a conventional MTT 3- 4,5-dimethylthiazol-2yl -2,5-diphenyltetrazolium bromide reduction assay. After 48 h exposure to six concentrations of Cu BrHAP 2 , cells were treated with MTT solution 2 mg/mL for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader Hidex, Turku, Finland . The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates.", "The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase LDH release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader.", "Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu BrHAP 2 was expressed as a percentage of the positive control. A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min.", "A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min. In brief, HT-29 cells 1 × 10 4 cells/well in 96-well plate were supplemented with Cu BrHAP 2 2 g/mL or DMSO negative control for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader Thermo Scientific . The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis.", "The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded.", "In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A 10 mg/mL and 50 L of propidium iodide PI 1 mg/mL were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer BD FACSCanto II . The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin .", "The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin . The plate was then supplemented with the working fluorescein solution 150 L , followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE .", "Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE . In brief, HT-29 cells 1 × 10 4 cells/mL were seeded in 96-well plates and treated with different concentrations of Cu BrHAP 2 and DMSO negative control for 24 h. After 30 min treatment with dihydroethidium DHE dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously .", "The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously . Plates with stained cells were analyzed using the ArrayScan HCS system Cellomics, PA, USA . Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit Promega, Madison, WI . HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme.", "HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. In brief, HT-29 cells 1.0 × 10 4 cells/well in a 96-well plate were treated with different concentrations of Cu BrHAP 2 for 3 h, followed by stimulation with TNF- 1 ng/mL for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate.", "After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends.", "All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends. An analysis of variance ANOVA was carried out using the prism statistical package GraphPad Software, USA . < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments.", "Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu BrHAP 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 .", "Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 . Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu BrHAP 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure.", "Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu BrHAP 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment Figure 3 . The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases.", "The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay.", "Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve AUC technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation.", "The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation. HT-29 cells were treated with different concentrations of Cu BrHAP 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation.", "As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu BrHAP 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells.", "Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway.", "To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor- TNF- . The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression .", "The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation . The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention.", "The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways . In the present study, the Cu BrHAP 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner.", "HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line CCD 841 showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu II compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation .", "Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry .", "Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis .", "This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis . As the main source of cellular ROS and adenosine triphosphate ATP , mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL.", "After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis.", "These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential MMP fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu BrHAP 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome .", "The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets .", "The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control.", "Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu BrHAP 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway." ]	1,607	5,284	Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper II complex on HT-29 colon cancer cells. The Cu BrHAP 2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC 50 value of 2.87 μg/ml after 72 h of treatment. HT-29 cells treated with Cu II complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G 1 cell population. At a concentration of 6.25 μg/ml, the Cu BrHAP 2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu II complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu BrHAP 2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents. Text: Cancer is a debilitating disease that afflicts a substantial portion of the world population in all generations and is a major health problem of global concern . Among the various types of cancer, colorectal cancer is the second and third most prevalent cancer among males and females in the United States, respectively. In spite of all the considerable progress in protective methods and recent improvements in screening techniques and chemotherapy, the 1-year and 5-year relative survival rates for patients suffering from colorectal cancer are 83.2% and 64.3%, respectively . In addition, due to bitter controversy over optimal methods for early detection, full compliance of patients with screening recommendations remains a major hindrance for diagnosis at the early stages of cancer development. Development of resistance to chemotherapy also represents a critical issue for which simultaneous treatment with various classes of therapeutics to reduce the resistance has yielded some success . Moreover, the numerous side effects of chemotherapeutic drugs on cancer patients, including hair loss, diarrhea, bleeding, and immunosuppression, have made the process 2 The Scientific World Journal of treatment more complicated . The highly regulated programmed cell death process of apoptosis is a matter of great interest in oncology and cancer therapy and represents a common molecular pathway for drug resistance and carcinogenesis . Maintenance of a constant cell number in the colonic mucosa is highly regulated through the balance between apoptosis and cell proliferation. The perturbation in this balance leads to an escape from normal cell number homeostasis and is associated with the progression of cancer cells . Thus, suppression of proliferation and elevation of apoptosis in these aberrant cells are suggested to be the essential mechanism for the inhibition of colon cancer. Furthermore, apoptosis and the factors involved in its mechanism of action also present a window that can be exploited for the improvement of potential therapeutic agents with high effectiveness and less adverse side effects . Hence, screening for novel compounds capable of inducing apoptosis in colon cancer cells that can be used alone or in combination with other chemotherapeutic drugs is a significant need and represents a critical challenge in medicinal chemistry. Metal complexes have been extensively utilized in clinics for centuries and have attracted numerous inorganic chemists to analyze them, with the main focus being medical applications . Copper, an essential trace element with an oxidative nature and bioessential activity in human metabolism, does not exist in an ionic form in biological systems. Thus, measurement of copper in the body is evaluated in the form of complexes with organic compounds . Schiff bases are a critical class of compounds in medical chemistry that have demonstrated significant chemotherapeutic and antibacterial application . Schiff base Cu II complexes revealed great potential for antiproliferative, antibacterial, and gastroprotective activity . This study evaluated the anticancer potential of a copper II complex derived from N,N -dimethyl ethylene diamine and 2-hydroxyacetophenone Schiff base ligand, Cu BrHAP 2 . Furthermore, the possible apoptotic mechanism underlying this activity was also examined. Dulbecco's Modified Eagle Medium DMEM, Life Technologies, Inc., Rockville, MD containing 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin G at 37 ∘ C in a humidified atmosphere of 5% CO 2 /95% air. The cells were plated at a fitting density in tissue culture flasks Corning, USA according to each experimental scale. Cell viability was measured by a conventional MTT 3- 4,5-dimethylthiazol-2yl -2,5-diphenyltetrazolium bromide reduction assay. After 48 h exposure to six concentrations of Cu BrHAP 2 , cells were treated with MTT solution 2 mg/mL for 2 h. The dark formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance was measured at 570 nm and 650 nm as a background using a microplate reader Hidex, Turku, Finland . The IC 50 value was determined as the concentration of Cu BrHAP 2 required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. All samples were prepared in triplicates. Assay. Measurement of lactate dehydrogenase LDH release is a biomarker for determining the cytotoxicity of a compound. Briefly, HT-29 cells were treated with different concentrations of Cu BrHAP 2 and Triton X-100 positive control for 48 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100 L of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. The amount of formazan salt and intensity of red color in treated and untreated samples were represented as the LDH activity of cells. The LDH release level in cells treated with Cu BrHAP 2 was expressed as a percentage of the positive control. A propidium iodide PI and acridine orange AO double staining assay were carried out for detection of apoptosis in the treated cells using a fluorescent microscope Leica attached with Q-Floro software according to a standard procedure. HT-29 cells 5 × 10 4 cells/mL in a 25 mL culture flask were plated, treated with Cu BrHAP 2 at the IC 50 concentration, and incubated for 24, 48, and 72 h. After harvesting the cells, they were stained with fluorescent dyes and observed under a UV-fluorescent microscope Olympus BX51 within 30 min. In brief, HT-29 cells 1 × 10 4 cells/well in 96-well plate were supplemented with Cu BrHAP 2 2 g/mL or DMSO negative control for 24 h. The live cells were then incubated with BrdU and Phospho-Histone H3 dyes for 30 min. After the cells were fixed and stained as described by the manufacturer's instructions, they were visualized and analyzed using the Cellomics ArrayScan HCS reader Thermo Scientific . The fluorescence intensities of the dyes were measured using a target activation bioapplication module. To confirm the result of the fluorescence cell cycle analysis, HT-29 cells 5 × 10 4 cells/mL were treated with Cu BrHAP 2 for 24, 48, and 72 h for flow cytometry analysis. After incubation, HT-29 cells were spun down at 1800 rpm for 5 min. Next, fixation of a cell population for flow cytometry analysis was carried out to restore integrity. In brief, the cell pellets were fixed by mixing them with 700 L of cold ethanol 90% and were then kept at 4 ∘ C overnight. Treated HT-29 cells were spun down, and the ethanol was discarded. After washing and suspending the cells in PBS, 25 L of RNase A 10 mg/mL and 50 L of propidium iodide PI 1 mg/mL were added to the fixed cells for 1 h at 37 ∘ C. The added RNase A limited the ability of PI to bind to only DNA molecules. At the end, the DNA content of the cells was analyzed by a flow cytometer BD FACSCanto II . The oxygen radical antioxidant capacity ORAC assay was carried out based on the protocols described in detail previously . In brief, Cu BrHAP 2 at the concentration of 100 g/mL was used for this assay in a total reaction volume of 200 L. The experiment was performed in a black 96-well microplate with 25 L of compound, blank solvent/PBS , standard trolox , or positive control quercetin . The plate was then supplemented with the working fluorescein solution 150 L , followed by a 5 min incubation at 37 ∘ . The total volume of 200 L was made up by adding 25 L of AAPH working solution. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm every 2 min for 2 h. The result was quantified by calculating the differences of area under the fluorescence decay curve AUC of samples and blank. The values were Trolox equivalents TE . In brief, HT-29 cells 1 × 10 4 cells/mL were seeded in 96-well plates and treated with different concentrations of Cu BrHAP 2 and DMSO negative control for 24 h. After 30 min treatment with dihydroethidium DHE dye, cells were fixed and washed with wash buffer as described by the manufacturer's instructions. In the presence of superoxides, DHE dye is oxidized to ethidium. The fluorescence intensity was determined by a fluorescent plate reader at an extension wavelength of 520 nm and an emission wavelength of 620 nm. The critical factors for monitoring the cell health, namely, cell loss, changes in cell permeability, cytochrome release, mitochondrial membrane potential changes, nuclear size, and morphological changes, were studied using a Cellomics Multiparameter Cytotoxicity 3 Kit as described in detail previously . Plates with stained cells were analyzed using the ArrayScan HCS system Cellomics, PA, USA . Caspases 3/7, -8, and 9 activities were determined using the commercial caspase-Glo 3/7, 8, and 9 assay kit Promega, Madison, WI . HT-29 cells 1.0 × 10 4 cells/well were seeded overnight in white-walled 96-well plates and treated with different concentrations of Cu BrHAP 2 for 24 h. According to the manufacturer's protocol, the treated cells were supplemented with caspase-Glo reagent 100 L and incubated at room temperature for 30 min. The active caspases from apoptotic cells caused the cleavage of aminoluciferin-labeled synthetic tetrapeptide, leading to the release of substrate for the luciferase enzyme. Caspase activities were analyzed using a Tecan Infinite 200 Pro Tecan, Männedorf, Switzerland microplate reader. In brief, HT-29 cells 1.0 × 10 4 cells/well in a 96-well plate were treated with different concentrations of Cu BrHAP 2 for 3 h, followed by stimulation with TNF- 1 ng/mL for 30 min. After discarding the medium, cells were fixed and stained using a Cellomics nucleus factor-B NF-B activation kit Thermo Scientific according to the manufacturer's instructions. Next, an Array Scan HCS Reader was used for evaluation of the plate. Cytoplasmic and nuclear NF-B intensity ratios were calculated using Cytoplasm to Nucleus Translocation Bioapplication software. The average intensity of 200 cells/well was determined. The ratios for untreated, treated, and TNF-stimulated cells were compared. All the experiments were performed at least three times independently. The results were presented as the mean ± standard deviation SD of the number of experiments shown in the legends. An analysis of variance ANOVA was carried out using the prism statistical package GraphPad Software, USA . < 0.05 was considered statistically significant. Cells of the Colon. Initially, the cytotoxicity of Cu BrHAP 2 was tested on HT-29 and CCD 841 cell lines. The IC 50 values of the Schiff base compound were determined based on the result collected from three independent MTT experiments. As indicated in Table 1 , Cu BrHAP 2 elicited a significant cytotoxicity and cell inhibitory effect after 24, 48, and 72 h of treatment on HT-29 cell. 2 -Induced LDH Release. Lactate dehydrogenase LDH release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of the Cu BrHAP 2 compound, as determined by the LDH release assay, was quantified on HT-29 cells treated with various concentrations of the Schiff base compound for 48 h. Cu BrHAP 2 induced a significant elevation in LDH release, demonstrating cytotoxicity at the 6.25 and 12.5 g/mL concentrations compared to the control cells Figure 2 . Microscopy and AO/PI Double Staining. Morphological changes in HT-29 cells treated with Cu BrHAP 2 compound were observed under a fluorescent microscope at 24, 48, and 72 h. The cells were scored under a fluorescent microscope to analyze viable cells, early apoptosis, and late apoptosis. Early apoptosis, defined as intervening AO within the fragmented DNA, was observed under bright green fluorescence. At the same time, control cells were visualized with a green intact nuclear structure. After 24 and 48 h of treatment with Cu BrHAP 2 , moderate apoptosis was observed in the form of blebbing and nuclear chromatin condensation. Furthermore, in the late stage of apoptosis, changes, such as the presence of a reddish-orange color due to binding of PI to denatured DNA, were observed after 72 h of treatment Figure 3 . The results showed that the Cu BrHAP 2 compound induced morphological features of apoptosis in a time-dependent manner. Figure 4 , demonstrated that there is no cell cycle arrest in the S/M phases. The lack of cell cycle arrest in the S/M phases suggested possible cell cycle arrest in the G 1 /G 2 phases. To determine the exact arrested phase, treated HT-29 cells were analyzed for cell cycle progression using flow cytometry. As expected, there was no significant arrest in the S/M phases. Meanwhile, significant cell cycle arrest in the G 1 phase was observed for HT-29 cells after 24 and 48 h of treatment Figure 5 . Assay. Antioxidant capacity was measured by ORAC assay, which is the only assay that involves the use of peroxyl radical as a prooxidant and quantifies activity via the area under the curve AUC technique. In our experiment, quercetin was used as a positive control. The result demonstrated that Cu BrHAP 2 exhibited low to moderate antioxidant activity compared to quercetin Table 2 . Formation. HT-29 cells were treated with different concentrations of Cu BrHAP 2 for 24 h and stained with DHE dye to determine the influence of the Schiff base compound on ROS production. The fluorescence intensities of DHE oxidization by ROS were quantified using a fluorescence microplate reader. As depicted in Figure 6 , exposure to the Schiff base compound caused a significant elevation in the ROS levels of treated HT-29 cells at the 6.25 g/mL concentration. To investigate the induction of apoptosis by Cu BrHAP 2 , nuclear morphological changes in HT-29 cells were analyzed by detection of nuclear condensation. As shown in Figure 7 , Hoechst 33342 staining demonstrated that nuclear condensation, which is directly related to apoptotic chromatin changes, emerged in some cells after treatment with Cu BrHAP 2 . Meanwhile, the permeability of treated cells was also elevated. Mitochondria are the main source for the production of ROS and adenosine triphosphate ATP and are critical in controlling the death and survival of cells. The reduction in fluorescence intensity depicted in Figure 6 Cu BrHAP 2 triggered the translocation of cytochrome from mitochondria into the cytosol during apoptosis in HT-29 cells. Activation. The elevation in ROS production associated with a collapse in MMP may lead to the activation of the caspase cascade. To investigate caspase activation, the bioluminescent intensities representing caspases 3/7, 8, and 9 activities were quantified in HT-29 cells treated with different concentrations of Cu BrHAP 2 for 24 h. As shown in Figure 8 , significant elevation in the activity of caspase-3/7 at the 6.25 g/mL concentration and caspase-9 at the 6.25 and 12.5 g/mL concentrations was observed in Cu BrHAP 2treated cells, while no significant change in the activity of caspase-8 was detected between treated and untreated HT-29 cells. Thus, the apoptosis induced by the Schiff base compound in HT-29 cells is possibly mediated via the intrinsic pathway, but not the extrinsic pathway. is a transcription factor that has a critical role in cytokine gene expression. NF-B activation and translocation to the nucleus to enable DNA-binding activity and facilitate target gene expression are mediated by inflammatory cytokines such as tumor necrosis factor- TNF- . The Cu BrHAP 2 Schiff base compound did not exhibit any inhibitory effect on translocation of TNF--stimulated NF-B in HT-29 treated cells, and TNF--stimulation led to NF-B translocation from the cytoplasm to the nucleus Figure 9 . Carcinogenesis is a multistage process in which unregulated cell proliferation as well as a reduction in apoptosis incidence serves as initial characterizations for its progression . One of the defense procedures in multicellular organisms is the destruction of undesirable cell development, which is defined as programmed cell death. Apoptosis is the most noticed programmed cell death mechanism and is characterized by distinct morphological changes such as membrane permeability, cell shrinkage, disruption of the mitochondrial membrane, and chromatin condensation . The disruption of cellular homeostasis between cell death and cell proliferation leads to cancer incidence , and agents that can induce apoptosis are known to have potential anticancer effects . Apoptosis pathways are effective targets for cancer therapy as well as chemoprevention. Numerous chemopreventive drugs have been determined to regulate key events or molecules in apoptosis-inducing signal transduction pathways . In the present study, the Cu BrHAP 2 Schiff base compound was evaluated for its ability to inhibit the growth of HT-29 cells using an MTT assay. HT-29 cells have recently been characterized as a suitable model for colon cancer studies . human colon cancer cells in a time-and dose-dependent manner. Meanwhile, the nontumorigenic colon cell line CCD 841 showed no cytotoxicity after treatment with the compound. The cytotoxic effect of the Cu II compound was also confirmed by measuring the level of LDH release from treated cells. Considerably elevated LDH release showed that the cytotoxicity of the Cu BrHAP 2 compound possibly occurred via the loss of membrane integrity, whether through activation of apoptosis or the necrosis pathway . The observation of early apoptosis and late apoptosis by fluorescent microscopy analysis and AO/PI double staining following treatment of HT-29 cells with the compound included some signs of apoptosis, namely, cytoplasmic shrinkage, membrane blebbing, and DNA fragmentation . We found that the number of cells with early apoptosis features was higher at earlier stages of treatment. However, when treatment time increased to 72 h, late apoptosis or necrosis characterizations were dominant among treated HT-29 cells. Concurrent detection of late apoptosis or necrosis is scientifically possible because treated HT-29 cells undergoing apoptosis may have progressed into necrosis due to the prolonged incubation with the Schiff base compound. To elucidate the mechanisms underlying the observed antiproliferative effect of the Cu II complex on cancer cells, cell cycle distribution was analyzed using BrdU and Phospho-Histone H3 staining along with flow cytometry . BrdU dye can attach to the synthesized DNA of replicating cells during the S phase of the cell cycle, while Phospho-Histone H3 dye stains cells in different mitotic stages. The cell cycle results from the BrdU and Phospho-Histone H3 double staining assay indicated that there were no significant changes in the number of cells in the S/M phases after the exposure of HT-29 cells to the Schiff base compound. This result suggests the possibility that the cells were arrested in the G 1 or G 2 phase of the cell cycle. Thus, the flow cytometry analysis of the cell cycle was performed to determine the exact arrested phase, and the results demonstrated significant cell cycle arrest at G 1 after 24 and 48 h of treatment, suggesting proliferative suppression via induction of apoptosis . Perturbation of mitochondrial membrane potential is one of the earliest intracellular events that occur following the induction of apoptosis . As the main source of cellular ROS and adenosine triphosphate ATP , mitochondria are the key regulators of mechanisms controlling the survival or death of cells. After confirming that the Cu BrHAP 2 Schiff base compound did not have significant antioxidant capacity in HT-29 cancer cells using the ORAC assay, the induction of ROS production in treated cells was analyzed. According to our study, after exposing the Cu II compound to HT-29 cells and analyzing the levels of ROS, it was demonstrated that the level of ROS in treated HT-29 cells was significantly elevated at a compound concentration of 6.25 g/mL. In metal-induced apoptosis, the mitochondria have the crucial role in mediating apoptosis through metal-induced ROS . The intrinsic or mitochondrial-dependent signaling pathway involves different factors of nonreceptor-mediated stimuli that induce intracellular signals. These signals, mainly through the p53 protein, act on the mitochondrialinitiated events. Excessive ROS production is a negative signal that can result in the failure of suppression of antiapoptotic factors, thereby triggering apoptosis. Therefore, we used mitochondrial membrane potential MMP fluorescent probes to examine the effect of elevated ROS production on the function of mitochondria in treated HT-29 cells. As shown in Figure 7 , changes in MMP after treatment with the Cu BrHAP 2 Schiff base compound leading to the membrane depolarization of the mitochondria were demonstrated by Rhodamine 123 release to the cytoplasm from the mitochondria matrix. The result implies that the induction of apoptosis by Cu II Schiff base complexes may be associated with the mitochondrial pathway . One of the important signals to initiate the procedure of apoptosis is cytosolic cytochrome . The release of cytochrome into the cytosol and reduction of its levels in the mitochondria have been shown to occur as a result of changes in MMP . As the result illustrated, the synthetic Schiff base compound also led to an increase in the level of cytochrome in the cytosol compared to the control. The excessive production of ROS from mitochondria and the collapse of MMP may activate the downstream caspase molecules and consequently lead to apoptotic cell death. After the binding of cytochrome to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation, which leads to active caspase-3/7, the most effective caspase with many cellular targets . In the extrinsic pathway, apoptosis is mediated by death receptors. As an example, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8 . Caspase-8 activation cleaves and activates downstream executioner caspases such as caspase-3/7 . In our study, the Cu BrHAP 2 schiff base compound induced significant elevation in the caspases 3/7 and 9 activities compared to the control. Meanwhile, there was no activation of caspase-8, suggesting that the apoptosis induced in HT-29 cells was mediated via the intrinsic mitochondrial pathway but not the extrinsic, death receptor-linked caspase-8 pathway. The supporting evidence of LDH release, ROS production, MMP suppression, elevation in the level of cytochrome , and activation of caspases 3/7 and 9 demonstrated the promising anticancer activity of the Cu BrHAP 2 Schiff base compound against the HT-29 colon cancer cell line via the intrinsic mitochondrial pathway.
What was the goal of this study?	to explore the feasibility of DNA vaccination of poultry	[ "BACKGROUND: Sustained outbreaks of highly pathogenic avian influenza HPAI H5N1 in avian species increase the risk of reassortment and adaptation to humans. The ability to contain its spread in chickens would reduce this threat and help maintain the capacity for egg-based vaccine production. While vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses. METHODOLOGY / PRINCIPAL FINDINGS: The ability of DNA vaccines encoding hemagglutinin HA proteins from different HPAI H5N1 serotypes was evaluated for its ability to elicit neutralizing antibodies and to protect against homologous and heterologous HPAI H5N1 strain challenge in mice and chickens after DNA immunization by needle and syringe or with a pressure injection device. These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs. The response was dose-dependent, and breadth was determined by the choice of the influenza virus HA in the vaccine.", "These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs. The response was dose-dependent, and breadth was determined by the choice of the influenza virus HA in the vaccine. Monovalent and trivalent HA vaccines were tested first in mice and conferred protection against lethal H5N1 A/Vietnam/1203/2004 challenge 68 weeks after vaccination. In chickens, protection was observed against heterologous strains of HPAI H5N1 after vaccination with a trivalent H5 serotype DNA vaccine with doses as low as 5 µg DNA given twice either by intramuscular needle injection or with a needle-free device. CONCLUSIONS/SIGNIFICANCE: DNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. In addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates.", "CONCLUSIONS/SIGNIFICANCE: DNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. In addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates. Text: The highly pathogenic H5N1 influenza virus causes lethal multi-organ disease in poultry, resulting in significant economic losses and a public health concern in many parts of the world. The greatest threats posed by this virus are its ability to cause mortality in humans, its potential to compromise food supplies, and its possible economic impacts. Viral maintenance in poultry potentiates the risk of human-to-human transmission and the emergence of a pandemic strain through reassortment. An effective, safe poultry vaccine that elicits broadly protective immune responses to evolving flu strains would provide a countermeasure to reduce the likelihood of transmission of this virus from domestic birds to humans and simultaneously would protect commercial poultry operations and subsistence farmers.", "Viral maintenance in poultry potentiates the risk of human-to-human transmission and the emergence of a pandemic strain through reassortment. An effective, safe poultry vaccine that elicits broadly protective immune responses to evolving flu strains would provide a countermeasure to reduce the likelihood of transmission of this virus from domestic birds to humans and simultaneously would protect commercial poultry operations and subsistence farmers. DNA vaccines have been shown to elicit robust immune responses in various animal species, from mice to nonhuman primates . In human trials, these vaccines elicit cellular and humoral immune responses against various infectious agents, including influenza, SARS, SIV and HIV. In addition to their ability to elicit antibody responses, they also stimulate antigenspecific and sustained T cell responses 6, 12, 13 . DNA vaccination has been used experimentally against various infectious agents in a variety of mammals, including cattle against infectious bovine rhinotracheitis/bovine diarrhea virus, leptospirosis and mycobacteriosis , pigs against classical swine fever virus and mycoplasmosis , and horses against West Nile virus and rabies .", "In addition to their ability to elicit antibody responses, they also stimulate antigenspecific and sustained T cell responses 6, 12, 13 . DNA vaccination has been used experimentally against various infectious agents in a variety of mammals, including cattle against infectious bovine rhinotracheitis/bovine diarrhea virus, leptospirosis and mycobacteriosis , pigs against classical swine fever virus and mycoplasmosis , and horses against West Nile virus and rabies . In addition, DNA vaccines have been tested against avian plasmodium infection in penguins and against influenza and infectious bursal disease in chickens , duck hepatitis B virus in ducks , and avian metapneumovirus and Chlamydia psittaci in turkeys reviewed in ref. . While they have been used in chickens to generate antisera to specific influenza viruses and confer protection against the low pathogenicity H5N2 strain , there is only one previous report of a monovalent DNA vaccine effective against H5N1 and that only against a matched H5N1 isolate ; no protection with multivalent DNA vaccines against heterologous strains has been reported. Development and characterization of a DNA vaccine modality for use in poultry offers a potential countermeasure against HPAI H5N1 avian influenza outbreaks.", "While they have been used in chickens to generate antisera to specific influenza viruses and confer protection against the low pathogenicity H5N2 strain , there is only one previous report of a monovalent DNA vaccine effective against H5N1 and that only against a matched H5N1 isolate ; no protection with multivalent DNA vaccines against heterologous strains has been reported. Development and characterization of a DNA vaccine modality for use in poultry offers a potential countermeasure against HPAI H5N1 avian influenza outbreaks. The virus can infect humans, typically from animal sources, including commercial and wild avian species, livestock, and possibly other non-domesticated animal species . While there is marked diversity in the host range of type A influenza viruses, many experts have speculated that a pandemic strain of type A influenza could evolve in avian species or avian influenza viruses could contribute virulent genes to a pandemic strain through reassortment . Thus, there is reason to consider vaccination of poultry that would stimulate potent and broad protective immune responses . In undertaking such efforts, it is important that there be a differentiation of infected from vaccinated animals so that animals can be protected and permit monitoring of new infections using proven and sensitive methodologies.", "Thus, there is reason to consider vaccination of poultry that would stimulate potent and broad protective immune responses . In undertaking such efforts, it is important that there be a differentiation of infected from vaccinated animals so that animals can be protected and permit monitoring of new infections using proven and sensitive methodologies. In this study, we used an automated high capacity needle-free injection device, Agro-JetH Medical International Technology, Inc., Denver, CO to explore the feasibility of DNA vaccination of poultry. After optimization of injection conditions, alternative multivalent DNA vaccine regimens were analyzed and compared for magnitude and breadth of neutralizing antibodies, as well as protective efficacy after challenge in mouse and chicken models of HPAI H5N1 infection. The findings suggest that it is possible to develop a multivalent DNA vaccine for poultry that can protect against multiple HPAI H5N1 strains and that could keep pace with the continued evolution of avian influenza viruses. Immunogenicity and neutralizing antibody specificity of alternative HA DNA vaccines in mice To evaluate the efficacy of multivalent DNA vaccines, initial studies were performed in mice.", "The findings suggest that it is possible to develop a multivalent DNA vaccine for poultry that can protect against multiple HPAI H5N1 strains and that could keep pace with the continued evolution of avian influenza viruses. Immunogenicity and neutralizing antibody specificity of alternative HA DNA vaccines in mice To evaluate the efficacy of multivalent DNA vaccines, initial studies were performed in mice. Expression vectors encoding HAs from ten phylogenetically diverse strains of influenza viruses were generated by synthesis of cDNAs see Materials and Methods in plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo . Animals were immunized with each expression vector intramuscularly IM at three week intervals, and the antisera were evaluated on day 14 after the third immunization for their ability to neutralize HPAI H5N1 pseudotyped lentiviral vectors as previously described . We have previously shown that lentiviral assay inhibition LAI yields similar results to microneutralization and HAI analyses with higher sensitivity in mice To determine whether immunization with multiple HAs simultaneously could expand the breadth of the neutralizing antibody response without significant loss of magnitude, a combination of 10 HA DNA vaccine immunogens was administered IM at proportionally lower concentration 1.5 mg per immunogen into groups of 10 mice see Materials and Methods . Remarkably, despite a log lower DNA concentration of each component, significant neutralizing antibody titers were generated to each of the 10 immunogens, with .80% neutralization against 6 out of 12 H5 HA pseudoviruses at dilutions of up to 1:400 Fig.", "We have previously shown that lentiviral assay inhibition LAI yields similar results to microneutralization and HAI analyses with higher sensitivity in mice To determine whether immunization with multiple HAs simultaneously could expand the breadth of the neutralizing antibody response without significant loss of magnitude, a combination of 10 HA DNA vaccine immunogens was administered IM at proportionally lower concentration 1.5 mg per immunogen into groups of 10 mice see Materials and Methods . Remarkably, despite a log lower DNA concentration of each component, significant neutralizing antibody titers were generated to each of the 10 immunogens, with .80% neutralization against 6 out of 12 H5 HA pseudoviruses at dilutions of up to 1:400 Fig. 2A . To evaluate whether similar breadth of immunity could be generated with fewer immunogens, two different combinations of 5 immunogens were selected, based on the phylogenetic diversity of HA among the avian influenza viruses and the crossreactivity of the neutralizing antibody responses of select individual immunogens Fig. 1 . As expected, there were substantial differences in the breadth of neutralization between these two sets of 5 immunogen multivalent vaccines Fig.", "1 . As expected, there were substantial differences in the breadth of neutralization between these two sets of 5 immunogen multivalent vaccines Fig. 2 , B vs. C . In one set, while neutralization of homologous strains was comparable to the monovalent and the 10 immunogen multivalent immune response, fewer cross-reactive antibodies were detected, directed most prominently against A/Iraq Protection of DNA-vaccinated mice against challenge with heterologous H5N1 A/Vietnam/1203/2004 influenza virus Mice immunized as described above were challenged with a heterologous H5N1 virus 68 weeks after the final DNA vaccination. Animals were then challenged with 10 LD 50 of the highly pathogenic A/Vietnam/1203/2004 virus intranasally, and morbidity and mortality were monitored for 21 days after the viral challenge. The control animals, injected with the plasmid expression vector with no insert, died within 10 days of infection.", "Animals were then challenged with 10 LD 50 of the highly pathogenic A/Vietnam/1203/2004 virus intranasally, and morbidity and mortality were monitored for 21 days after the viral challenge. The control animals, injected with the plasmid expression vector with no insert, died within 10 days of infection. Complete survival was observed in the groups immunized with the 10 component and set 2 of the 5 component multivalent DNA vaccines Fig. 3 . Immunization with HA derived from the A/ Indonesia/05/2005 strain or set 1 of the 5 component multivalent DNA vaccine showed a survival rate approaching 90%. In contrast, animals injected with HA plasmid DNA derived from A/ Anhui/1/2005, which has diverged more from A/Vietnam/ 1203/2004, showed a lower percent survival 70% after lethal viral challenge.", "Immunization with HA derived from the A/ Indonesia/05/2005 strain or set 1 of the 5 component multivalent DNA vaccine showed a survival rate approaching 90%. In contrast, animals injected with HA plasmid DNA derived from A/ Anhui/1/2005, which has diverged more from A/Vietnam/ 1203/2004, showed a lower percent survival 70% after lethal viral challenge. Survival differences between groups were assessed using a log-rank test and the Gehan-Wilcoxon test on the survival curves for pairs of groups. A test was deemed significant if the pvalue was ,0.01. Mice injected IM with different HAs, A/ Indonesia/5/05, A/Anhui/1/05, 10HA, 5 HA Set 1 , or 5 HA Set 2 showed a significant difference compared to control all p values,0.001 . Among the HA-immunized groups, there was no significant difference between any two groups p.0.08 for all comparisons .", "Mice injected IM with different HAs, A/ Indonesia/5/05, A/Anhui/1/05, 10HA, 5 HA Set 1 , or 5 HA Set 2 showed a significant difference compared to control all p values,0.001 . Among the HA-immunized groups, there was no significant difference between any two groups p.0.08 for all comparisons . For example, no significant difference was observed between the A/Anhui/1/05 group, which had the least survival among the HA immunized groups 7 out of 10 , and other HA groups: A/Indonesia/5/05 p = 0.377 , 10 HA p = 0.082 , 5 HA Set 1 p = 0.101 , or 5 HA Set 2 p = .411 . Therefore, we cannot exclude the possibility that the 3 deaths in the A/Anhui/1/05 group may have been due to random chance. Since it is desirable to confer protective immunity in poultry and HA DNA vaccination was effective in mice, we next examined the breadth and potency of single or multiple HA plasmid immunization in chickens. The ability of chickens to generate specific antibodies was assessed with three strains that showed broad cross protection in mouse studies A/Vietnam/1203/2004, A/Anhui/ 1/2005 and A/Indonesia/05/2005 , administered individually or in combination, by different injection methods.", "Since it is desirable to confer protective immunity in poultry and HA DNA vaccination was effective in mice, we next examined the breadth and potency of single or multiple HA plasmid immunization in chickens. The ability of chickens to generate specific antibodies was assessed with three strains that showed broad cross protection in mouse studies A/Vietnam/1203/2004, A/Anhui/ 1/2005 and A/Indonesia/05/2005 , administered individually or in combination, by different injection methods. In addition to needle injection, a needle-free repetitive injection device, Agro-JetH Medical International Technology, Inc., Denver, CO , was analyzed. This device disperses the 0.1 to 5 ml injection doses into the dermal, subcutaneous, or intramuscular tissue depending upon the pressure adjustments, powered by a CO 2 gas pressure plunger . The injection conditions were determined by histologic analysis of tissues that received injections of India ink; a pressure of 48 psi was chosen since it enabled consistent delivery into intradermal and subcutaneous tissues Fig. S1 .", "The injection conditions were determined by histologic analysis of tissues that received injections of India ink; a pressure of 48 psi was chosen since it enabled consistent delivery into intradermal and subcutaneous tissues Fig. S1 . Immunization of chickens with the control plasmid CMV/R without an HA gene insert elicited minimal neutralizing antibody titers compared to HA-immunized animals 1 week after 3 DNA immunizations. Nearly all chickens immunized with either monovalent or multivalent HA DNA vaccines generated significant neutralization titers Fig. 4 and Table S1 . In general, there was a progressive increase in the amount of neutralization after each successive DNA vaccination data not shown with maximal response at 1 week after the 3 rd DNA immunization, with highest and most consistent levels in the trivalent vaccine group delivered with the Agro-JetH device.", "4 and Table S1 . In general, there was a progressive increase in the amount of neutralization after each successive DNA vaccination data not shown with maximal response at 1 week after the 3 rd DNA immunization, with highest and most consistent levels in the trivalent vaccine group delivered with the Agro-JetH device. Neutralization of the Indonesia HA strain was the most robust, with neutralization nearing 100% at titers greater than 1:3200. Both the monovalent and multivalent vaccines elicited robust homologous vaccine Fig. 4 . Even though one chicken .", "Both the monovalent and multivalent vaccines elicited robust homologous vaccine Fig. 4 . Even though one chicken . in the multivalent vaccine group produced almost the same degree of neutralization at each time point and was protected, it did not produce a high neutralizing antibody titer for reasons that were uncertain but possibly related to a non-specific inhibitor in the sera. To determine whether chickens immunized with single or multiple DNA vaccines were protected from a lethal challenge of a heterologous HPAI H5N1 virus, vaccinated chickens were In panels B and C, mice n = 10 were immunized with 15 mg of plasmid 3 mg each three times at 3 week intervals. Serum pools from the immunized animals were collected 14 days after the third immunization.", "To determine whether chickens immunized with single or multiple DNA vaccines were protected from a lethal challenge of a heterologous HPAI H5N1 virus, vaccinated chickens were In panels B and C, mice n = 10 were immunized with 15 mg of plasmid 3 mg each three times at 3 week intervals. Serum pools from the immunized animals were collected 14 days after the third immunization. The antisera were tested against the 12 indicated pseudotyped lentiviral vectors at varying dilutions. Error bars at each point indicate the standard deviation; each sample was evaluated in triplicate. In general, the immunized serum neutralized all tested pseudotyped lentiviruses at low dilutions while differences were often observed at high dilution. .1371/journal.pone.0002432.g002 inoculated with 20 LD 50 of highly pathogenic A/Vietnam/1203/ 2004 heterologous virus intranasally using standard methods and monitored for morbidity, mortality, viral shedding and serum antibodies.", "In general, the immunized serum neutralized all tested pseudotyped lentiviruses at low dilutions while differences were often observed at high dilution. .1371/journal.pone.0002432.g002 inoculated with 20 LD 50 of highly pathogenic A/Vietnam/1203/ 2004 heterologous virus intranasally using standard methods and monitored for morbidity, mortality, viral shedding and serum antibodies. While all the control animals died within 2 days of infection, 100% survival was noted in the rest of the chickens Fig. 5A . The animals that were healthy, showing no signs of clinical disease or malaise, were euthanized on day 14. There was no evidence for viral shedding monitored via tracheal and cloacal swabs of infected chickens 2-14 days after challenge as determined by embryonal inoculation data not shown: egg infectious dose 50 EID 50 limit of detection ,100 virus particles .", "The animals that were healthy, showing no signs of clinical disease or malaise, were euthanized on day 14. There was no evidence for viral shedding monitored via tracheal and cloacal swabs of infected chickens 2-14 days after challenge as determined by embryonal inoculation data not shown: egg infectious dose 50 EID 50 limit of detection ,100 virus particles . To compare the relative efficacy of DNA vaccines delivered IM by needle and syringe versus the needle-free Agro-JetH device injection, a dose-response study was performed with amounts of DNA vaccine ranging from 500 to 0.5 mg with two inoculations. In these experiments, the HA derived from A/chicken/Nigeria/641/ 2006 was substituted for A/Vietnam/1203/2004 since it represented a more contemporary isolate. The observed rate of protection was higher among the animals receiving 5 mg by Agro-Jet 8/8 than by IM injection 6/8 Fig. 5, B vs. C .", "The observed rate of protection was higher among the animals receiving 5 mg by Agro-Jet 8/8 than by IM injection 6/8 Fig. 5, B vs. C . Both modes provided complete protection for all animals at doses higher than this, and 25% protection for the animals receiving 0.5 mg doses Fig. 5B, C . Survival differences between consecutive doses were assessed using a log-rank test on the survival curves for pairs of groups. A test was deemed significant if the p-value was ,.01, and marginally significant if the p-value was ,.05 but ..01. Chickens injected IM showed a marginally significant difference between 0.5 and 5 mg p = .047 .", "A test was deemed significant if the p-value was ,.01, and marginally significant if the p-value was ,.05 but ..01. Chickens injected IM showed a marginally significant difference between 0.5 and 5 mg p = .047 . In the same group there was a significant difference between control and 5, 50 and 500 mg p,.001 for all comparisons and the difference between control and 0.5 mg was marginally significant p = .016 . Chickens that were injected using Agro-JetH showed a significant difference between 0.5 and 5 mg p = .004 and between control and 5, 50, and 500 mg p,.001 for all comparisons . There were no differences between control and 0.5 mg or between 5, 50, and 500 mg. Lastly, the survival differences between Agro-JetH and IM for each dose group were not significant. The neutralizing antibody response to homologous and heterologous HAs corresponded with protection and correlated with dose, with higher titers elicited by injection with Agro-JetH compared to needle Table S2 .", "There were no differences between control and 0.5 mg or between 5, 50, and 500 mg. Lastly, the survival differences between Agro-JetH and IM for each dose group were not significant. The neutralizing antibody response to homologous and heterologous HAs corresponded with protection and correlated with dose, with higher titers elicited by injection with Agro-JetH compared to needle Table S2 . We assessed viable viral shedding after inoculation by chick embryo inoculation three days after virus challenge Week 8 . While we noted some embryonic lethality at the 0.5 mg dose, there was no embryonic lethality at 5, 50 or 500 mg groups data not shown . Since the HPAI H5N1 virus first appeared ten years ago, this highly pathogenic avian influenza virus has shown increasing diversification and dissemination in Asia, Africa, and Europe 28, . In addition to its effects on human health by crossspecies transmission and ability to compromise food sources, it poses a continuing threat to public health as it evolves and adapts in different species.", "Since the HPAI H5N1 virus first appeared ten years ago, this highly pathogenic avian influenza virus has shown increasing diversification and dissemination in Asia, Africa, and Europe 28, . In addition to its effects on human health by crossspecies transmission and ability to compromise food sources, it poses a continuing threat to public health as it evolves and adapts in different species. The pandemic potential of this virus, especially as it relates to the poultry industry and for reservoir avian hosts, underscores the need for a vaccine that offers broad spectrum immunity and protection against lethal viral challenge. While the virus remains restricted in its ability to infect humans and undergo efficient human-to-human transmission , its persistence and spread in poultry increases the risk of the emergence of a pandemic strain. One approach to pandemic risk reduction is to limit the propagation of the virus in poultry and other relevant avian species. We have previously reported that DNA vaccines encoding HA can confer protection against a highly lethal human pandemic influenza virus, the 1918 H1N1 virus, in mice .", "One approach to pandemic risk reduction is to limit the propagation of the virus in poultry and other relevant avian species. We have previously reported that DNA vaccines encoding HA can confer protection against a highly lethal human pandemic influenza virus, the 1918 H1N1 virus, in mice . DNA vaccines offer several advantages, including the ability to express diverse antigens, tolerability in various hosts, ease of delivery, and stability for storage and distribution without the necessity of maintaining a cold chain; they have been shown to be safe and efficacious in a variety of animal models . Because they do not contain other viral proteins used to screen for infection, they also address the need to differentiate vaccinated from infected animals. There is evidence that DNA vaccination elicits cell-mediated immunity against influenza HA in addition to inducing an antibody response , an effect that could significantly contribute to protective immunity as viruses show genetic drift and reduced susceptibility to neutralization. Ideally, a highly effective influenza vaccine should not only be able to let the host develop a protective immune response against a matching live virus challenge but also elicit robust protective immune responses against a broad range of homologous and heterologous H5 influenza strains.", "There is evidence that DNA vaccination elicits cell-mediated immunity against influenza HA in addition to inducing an antibody response , an effect that could significantly contribute to protective immunity as viruses show genetic drift and reduced susceptibility to neutralization. Ideally, a highly effective influenza vaccine should not only be able to let the host develop a protective immune response against a matching live virus challenge but also elicit robust protective immune responses against a broad range of homologous and heterologous H5 influenza strains. A multivalent H5 vaccine containing diverse serotypes could expand the antigenic breadth sufficiently to provide protection against heterologous challenge and may preclude the emergence of vaccine-resistant strains that may arise due to evolutionary vaccine pressure on the virus. Due to the antigenic drift and shift of the influenza virus genome, it has been very difficult to predict the next dominant strain of an avian endemic outbreak. DNA vaccines can be synthesized in a relatively short period of time, and the targeted mutations can be tailored to specific viral serotypes. The mutations promote a focused and enhanced immune response that may be particularly important in the event of an outbreak where specificity is the key to epidemic control.", "DNA vaccines can be synthesized in a relatively short period of time, and the targeted mutations can be tailored to specific viral serotypes. The mutations promote a focused and enhanced immune response that may be particularly important in the event of an outbreak where specificity is the key to epidemic control. The use of modified codons ensures maximal expression in the host and eliminates the possibility of recombination with influenza viruses that might potentially generate new strains. A more broadly protective murine vaccine was developed here by including more HAs from varying strains in the multivalent vaccine Figs. 2 and 3 . However, it is less practical to include large numbers of different HAs in one vaccine due to the cost and complexity of manufacturing such a vaccine.", "2 and 3 . However, it is less practical to include large numbers of different HAs in one vaccine due to the cost and complexity of manufacturing such a vaccine. Therefore, we simplified the vaccine regimen based on cross-neutralization studies and phylogenetic relationships. A trivalent vaccine was subsequently identified for further studies. Due . While three DNA immunizations were used initially to demonstrate protective immunity and have been used previously to elicit protection in mice , we found that effective protective immunity could be induced with two DNA vaccinations and as little as 5 mg trivalent DNA immunization using the ID/SC route with the Agro-JetH device.", "Due . While three DNA immunizations were used initially to demonstrate protective immunity and have been used previously to elicit protection in mice , we found that effective protective immunity could be induced with two DNA vaccinations and as little as 5 mg trivalent DNA immunization using the ID/SC route with the Agro-JetH device. In addition, based on the chick embryo inoculation data, we believe that there is effective neutralization of the virus and lack of infectious viral shedding in chicken vaccinated with as little as 5 mg of DNA. The device's capacity for rapid repetitive injection and the lower quantity and stability of DNA enhance the practicality and utility of this approach for vaccination of endangered species in captivity or administration to poultry or other animals. A/Vietnam/1203/2004 H5N1 A/VN/1203/04 was obtained from the repository at the Centers for Disease Control and Prevention CDC , Atlanta, Georgia. The virus was propagated in 10-day old embryonated chicken eggs at 35uC and stored at 270uC until use.", "A/Vietnam/1203/2004 H5N1 A/VN/1203/04 was obtained from the repository at the Centers for Disease Control and Prevention CDC , Atlanta, Georgia. The virus was propagated in 10-day old embryonated chicken eggs at 35uC and stored at 270uC until use. The virus was titrated by the Reed and Muench method to determine EID 50 . GenBank ABD28180 were synthesized using human-preferred codons GeneArt, Regensburg, Germany . HA cDNAs from diverse strains of influenza viruses were then inserted into plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo . For initial trivalent immunizations in chickens, the A/Vietnam/1203/ 2004, A/Anhui/1/2005 and A/Indonesia/05/2005 strains were used and in the dose response study, the Vietnam strain was replaced with A/chicken/Nigeria/641/2006.", "HA cDNAs from diverse strains of influenza viruses were then inserted into plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo . For initial trivalent immunizations in chickens, the A/Vietnam/1203/ 2004, A/Anhui/1/2005 and A/Indonesia/05/2005 strains were used and in the dose response study, the Vietnam strain was replaced with A/chicken/Nigeria/641/2006. The immunogens used in DNA vaccination contained a cleavage site mutation PQRERRRKKRG to PQRETRG as previously described . This mutation was generated by site-directed mutagenesis using a QuickChange kit Stratagene, La Jolla, CA . DNA immunization of mice week old female BALB/c mice were purchased from The Jackson Laboratory and maintained in the AAALAC-accredited Vaccine Research Center Animal Care Facility Bethesda, MD under specific pathogen-free conditions. All experiments were approved by the Vaccine Research Center Animal Care and Use Committee.", "DNA immunization of mice week old female BALB/c mice were purchased from The Jackson Laboratory and maintained in the AAALAC-accredited Vaccine Research Center Animal Care Facility Bethesda, MD under specific pathogen-free conditions. All experiments were approved by the Vaccine Research Center Animal Care and Use Committee. The mice were immunized as previously described . Briefly, mice 10 animals for all test groups, 20 animals for the The study was carried out in the AAALAC-accredited animal facility at the University of Maryland School of Medicine. Six groups of 8 one-day-old male and female SPAFAS White Leghorn Chickens, Gallus domesticus, were obtained from Charles River Laboratories Connecticut . The animals were housed in brooder and grower cages McMurray Hatcheries, Iowa .", "Six groups of 8 one-day-old male and female SPAFAS White Leghorn Chickens, Gallus domesticus, were obtained from Charles River Laboratories Connecticut . The animals were housed in brooder and grower cages McMurray Hatcheries, Iowa . Feed Teklad Japanese Quail Diet -3050, Harlan-Teklad, WI and water were provided to the animals ad libitum. The study was performed in strict accordance with the ''Guide'' after approvals from the Animal Care and Use Committees of the Vaccine Research Center, NIH and the University of Maryland. DNA immunizations were performed as described at 0, 3 and 6 weeks. A total dose of 500 mg of one or a combination of the following DNA plasmids in a volume of 250 ml was administered to each animal: pCMV/ R, pCMV/R-HA Agro-JetH is a needle-free device used for mass delivery of vaccines and drugs in livestock and poultry.", "DNA immunizations were performed as described at 0, 3 and 6 weeks. A total dose of 500 mg of one or a combination of the following DNA plasmids in a volume of 250 ml was administered to each animal: pCMV/ R, pCMV/R-HA Agro-JetH is a needle-free device used for mass delivery of vaccines and drugs in livestock and poultry. The device is semiautomatic and requires a small CO 2 tank or compressed air for low pressure delivery. Upon trigger activation, CO 2 disperses the injectate at a precise dose into the muscle, dermis or subcutaneous tissue depending on the setting that was standardized for our use. We used an effective volume of 0.1 ml in our injectate . In this study we were able to effectively deliver 0.1 ml of injectate into the animal's dermis/subcutaneous tissue at a pressure of 48-55 psi.", "We used an effective volume of 0.1 ml in our injectate . In this study we were able to effectively deliver 0.1 ml of injectate into the animal's dermis/subcutaneous tissue at a pressure of 48-55 psi. Sixty-eight weeks after the last immunization, female BALB/c mice were lightly anesthetized with Ketamine/Xylazine and inoculated intranasally with 10 LD 50 of A/Vietnam/1203/2004 virus diluted in phosphate-buffered saline in a 50 ul volume. Mice were monitored daily for morbidity and measured for weight loss and mortality for 21 days post infection. Any mouse that had lost more than 25% of its body weight was euthanized. All experiments involving the HPAI virus were conducted in an AAALAC accredited facility BioQual Inc., Gaithersburg, MD under BSL 3 conditions that included enhancements required by the USDA and the Select Agent Program.", "Any mouse that had lost more than 25% of its body weight was euthanized. All experiments involving the HPAI virus were conducted in an AAALAC accredited facility BioQual Inc., Gaithersburg, MD under BSL 3 conditions that included enhancements required by the USDA and the Select Agent Program. White Leghorn chickens were challenged one week after the last immunization with 20 lethal dose 50 LD 50 of A/Vietnam/1203/04 H5N1 influenza A virus, equivalent to 2610 4 EID 50 based on previous challenges . Chickens were infected with 200 ml virus intranasally. Tracheal and cloacal swabs were collected days 3 and 5 post-challenge and stored in glass vials containing BHI medium BBL TM Brain Heart Infusion, Becton Dickinson at 280uC. Blood was collected 14 days post-challenge and serum was titered by microneutralization assay.", "Tracheal and cloacal swabs were collected days 3 and 5 post-challenge and stored in glass vials containing BHI medium BBL TM Brain Heart Infusion, Becton Dickinson at 280uC. Blood was collected 14 days post-challenge and serum was titered by microneutralization assay. Chickens were observed and scored daily for clinical signs of infection, morbidity and mortality. Chickens that survived the study were bled and humanely euthanized at day 14 post-challenge. Lungs, heart, intestine and kidney were collected and samples were stored in formalin for histopathology. Experiments were carried out under BSL3+ conditions with investigators wearing appropriate protective equipment and compliant with all Institutional Animal Care and Use Committee-approved protocols and under Animal Welfare Act regulations at the University of Maryland, College Park, Maryland.", "Lungs, heart, intestine and kidney were collected and samples were stored in formalin for histopathology. Experiments were carried out under BSL3+ conditions with investigators wearing appropriate protective equipment and compliant with all Institutional Animal Care and Use Committee-approved protocols and under Animal Welfare Act regulations at the University of Maryland, College Park, Maryland. Representative tracheal and cloacal swabs were chosen to run an EID 50 assay for comparison and virus titers were determine by the method of Reed and Meunch . Briefly, swabs were used to infect 10 day-old embryonated chicken eggs in 10-fold dilutions. Three eggs were inoculated per dilution and incubated for 48 hours before titration. Neutralizing antibodies were titrated from serum samples collected week 5 and 7 post-vaccination and day 14 post-challenge.", "Three eggs were inoculated per dilution and incubated for 48 hours before titration. Neutralizing antibodies were titrated from serum samples collected week 5 and 7 post-vaccination and day 14 post-challenge. The microneutralization assay was performed using a 96-well plate format. Serum was treated with receptor-destroying enzyme Denka Seiken Co. and treated at 37uC per the manufacturer's instructions. After an overnight incubation and subsequent inactivation samples were brought to a final dilution of 1:10 using PBS and each sample was serially diluted and virus, diluted to 100 TCID 50 , was added to each well. The plates were then incubated at 37uC, 5% CO 2 for 1-2 hours.", "After an overnight incubation and subsequent inactivation samples were brought to a final dilution of 1:10 using PBS and each sample was serially diluted and virus, diluted to 100 TCID 50 , was added to each well. The plates were then incubated at 37uC, 5% CO 2 for 1-2 hours. Following incubation, supernatants were used to infect a second 96-well plate of MDCK cells. Microplates were incubated at 4uC for 15 minutes and then 37uC, 5% CO 2 for 45 minutes. Supernatants of serum and virus were then discarded and 200 ml of OptiMEM containing 1X antibiotics/antimycotics, 1 mg/ml TPCK-trypsin was added and incubated at 37uC, 5% CO 2 for 3 days. After 3 days, 50 ml of the supernatant from each well was transferred into a new 96-well microplate, and an HA assay was performed to calculate the antibody titers.", "Supernatants of serum and virus were then discarded and 200 ml of OptiMEM containing 1X antibiotics/antimycotics, 1 mg/ml TPCK-trypsin was added and incubated at 37uC, 5% CO 2 for 3 days. After 3 days, 50 ml of the supernatant from each well was transferred into a new 96-well microplate, and an HA assay was performed to calculate the antibody titers. Virus and cell controls were included in the assay. Two-fold dilutions of heat-inactivated sera were tested in a microneutralization assay as previously described for the presence of antibodies that neutralized the infectivity of 100 TCID 50 50% tissue culture infectious dose of the A/Vietnam/ 1203/2004 H5N1 virus on MDCK cell monolayers by using two wells per dilution on a 96-well plate. The recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described . For the neutralization assay, antisera from immunized animals were heat-inactivated at 55uC for 30 minutes and mixed with 50 ml of pseudovirus at various dilutions.", "The recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described . For the neutralization assay, antisera from immunized animals were heat-inactivated at 55uC for 30 minutes and mixed with 50 ml of pseudovirus at various dilutions. The sera/virus mixture was then added to 293A cells in 96-well B&W TC Isoplates Wallac, Turku, Finland; 12,000 cells/well . Two hours later, the plates were washed and fresh medium was added. Cells were lysed in mammalian cell lysis buffer Promega, Madison, WI 24 hrs after infection and luciferase activity was measured using the Luciferase Assay System Promega, Madison, WI . The following strains were used for the production of pseudotyped viruses: for HA we used A/Thailand/1 KAN- The HA/HI titers were determined as previously described .", "Cells were lysed in mammalian cell lysis buffer Promega, Madison, WI 24 hrs after infection and luciferase activity was measured using the Luciferase Assay System Promega, Madison, WI . The following strains were used for the production of pseudotyped viruses: for HA we used A/Thailand/1 KAN- The HA/HI titers were determined as previously described . Briefly, HA titers were calculated using 50 ml of 0.5% chicken red blood cell suspension in PBS added to 50 ml of twofold dilutions of virus in PBS. This mix was incubated at room temperature for 30 minutes. The HA titers were calculated as the reciprocal value of the highest dilution that caused complete hemagglutination. HI titers were calculated by titrating 50 ml of antiserum treated with receptor-destroying enzyme and an equivalent amount of A/Vietnam/1203/2004 virus four hemagglutinating doses was added to each well.", "The HA titers were calculated as the reciprocal value of the highest dilution that caused complete hemagglutination. HI titers were calculated by titrating 50 ml of antiserum treated with receptor-destroying enzyme and an equivalent amount of A/Vietnam/1203/2004 virus four hemagglutinating doses was added to each well. Wells were incubated at room temperature for 30 minutes and 50 ml of a 0.5% suspension of chicken red blood cells was added. HI titers were calculated after 30 minutes as the reciprocal of the serum dilution that inhibited hemagglutination. Table S1 Hemagglutination inhibition HI , microneutralization titer NT , and LAI of sera from individual chickens immunized with different vaccines. Sera from immunized animals were obtained at week 5 or 7, a week before or after the final boost, and neutralization was assessed by HI, microneutralization NT and LAI shown as IC 50 .", "Table S1 Hemagglutination inhibition HI , microneutralization titer NT , and LAI of sera from individual chickens immunized with different vaccines. Sera from immunized animals were obtained at week 5 or 7, a week before or after the final boost, and neutralization was assessed by HI, microneutralization NT and LAI shown as IC 50 . Individual animal serum of each group is shown and was analyzed as described in the Materials and Methods section. Figure S1 Characterization of needle-free Agro-JetH DNA immunization in chickens. To evaluate the distribution of fluid into superficial or deep layers of subcutaneous tissues after delivery by AgroJetH, 4 or 7 week old chickens were injected with a solution containing India ink with this needle-free device at various pressures, ranging from 45 to 55 mm Hg. Three sites thigh, wing and breast were used, and biopsies were taken for routine hematoxylin and eosin staining.", "To evaluate the distribution of fluid into superficial or deep layers of subcutaneous tissues after delivery by AgroJetH, 4 or 7 week old chickens were injected with a solution containing India ink with this needle-free device at various pressures, ranging from 45 to 55 mm Hg. Three sites thigh, wing and breast were used, and biopsies were taken for routine hematoxylin and eosin staining. Representative sections of thigh injections are shown from 7 week old chickens and were similar at 4 weeks data not shown . While the 48 mm Hg pressure deposited the injectate into the dermis/subcutaneous region left , the higher pressure injections, 52 and 58 mm Hg, deposited the injectate into the subcutaneous and muscle layers middle, right . 48 mm Hg consistently provided an optimal pressure to deposit the injectate into the dermis and subcutaneous tissue and was chosen for all AgroJetH immunizations. Found at: .1371/journal.pone.0002432.s003 10.74 MB DOC" ]	1,608	5,285	BACKGROUND: Sustained outbreaks of highly pathogenic avian influenza HPAI H5N1 in avian species increase the risk of reassortment and adaptation to humans. The ability to contain its spread in chickens would reduce this threat and help maintain the capacity for egg-based vaccine production. While vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses. METHODOLOGY / PRINCIPAL FINDINGS: The ability of DNA vaccines encoding hemagglutinin HA proteins from different HPAI H5N1 serotypes was evaluated for its ability to elicit neutralizing antibodies and to protect against homologous and heterologous HPAI H5N1 strain challenge in mice and chickens after DNA immunization by needle and syringe or with a pressure injection device. These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs. The response was dose-dependent, and breadth was determined by the choice of the influenza virus HA in the vaccine. Monovalent and trivalent HA vaccines were tested first in mice and conferred protection against lethal H5N1 A/Vietnam/1203/2004 challenge 68 weeks after vaccination. In chickens, protection was observed against heterologous strains of HPAI H5N1 after vaccination with a trivalent H5 serotype DNA vaccine with doses as low as 5 µg DNA given twice either by intramuscular needle injection or with a needle-free device. CONCLUSIONS/SIGNIFICANCE: DNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. In addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates. Text: The highly pathogenic H5N1 influenza virus causes lethal multi-organ disease in poultry, resulting in significant economic losses and a public health concern in many parts of the world. The greatest threats posed by this virus are its ability to cause mortality in humans, its potential to compromise food supplies, and its possible economic impacts. Viral maintenance in poultry potentiates the risk of human-to-human transmission and the emergence of a pandemic strain through reassortment. An effective, safe poultry vaccine that elicits broadly protective immune responses to evolving flu strains would provide a countermeasure to reduce the likelihood of transmission of this virus from domestic birds to humans and simultaneously would protect commercial poultry operations and subsistence farmers. DNA vaccines have been shown to elicit robust immune responses in various animal species, from mice to nonhuman primates . In human trials, these vaccines elicit cellular and humoral immune responses against various infectious agents, including influenza, SARS, SIV and HIV. In addition to their ability to elicit antibody responses, they also stimulate antigenspecific and sustained T cell responses 6, 12, 13 . DNA vaccination has been used experimentally against various infectious agents in a variety of mammals, including cattle against infectious bovine rhinotracheitis/bovine diarrhea virus, leptospirosis and mycobacteriosis , pigs against classical swine fever virus and mycoplasmosis , and horses against West Nile virus and rabies . In addition, DNA vaccines have been tested against avian plasmodium infection in penguins and against influenza and infectious bursal disease in chickens , duck hepatitis B virus in ducks , and avian metapneumovirus and Chlamydia psittaci in turkeys reviewed in ref. . While they have been used in chickens to generate antisera to specific influenza viruses and confer protection against the low pathogenicity H5N2 strain , there is only one previous report of a monovalent DNA vaccine effective against H5N1 and that only against a matched H5N1 isolate ; no protection with multivalent DNA vaccines against heterologous strains has been reported. Development and characterization of a DNA vaccine modality for use in poultry offers a potential countermeasure against HPAI H5N1 avian influenza outbreaks. The virus can infect humans, typically from animal sources, including commercial and wild avian species, livestock, and possibly other non-domesticated animal species . While there is marked diversity in the host range of type A influenza viruses, many experts have speculated that a pandemic strain of type A influenza could evolve in avian species or avian influenza viruses could contribute virulent genes to a pandemic strain through reassortment . Thus, there is reason to consider vaccination of poultry that would stimulate potent and broad protective immune responses . In undertaking such efforts, it is important that there be a differentiation of infected from vaccinated animals so that animals can be protected and permit monitoring of new infections using proven and sensitive methodologies. In this study, we used an automated high capacity needle-free injection device, Agro-JetH Medical International Technology, Inc., Denver, CO to explore the feasibility of DNA vaccination of poultry. After optimization of injection conditions, alternative multivalent DNA vaccine regimens were analyzed and compared for magnitude and breadth of neutralizing antibodies, as well as protective efficacy after challenge in mouse and chicken models of HPAI H5N1 infection. The findings suggest that it is possible to develop a multivalent DNA vaccine for poultry that can protect against multiple HPAI H5N1 strains and that could keep pace with the continued evolution of avian influenza viruses. Immunogenicity and neutralizing antibody specificity of alternative HA DNA vaccines in mice To evaluate the efficacy of multivalent DNA vaccines, initial studies were performed in mice. Expression vectors encoding HAs from ten phylogenetically diverse strains of influenza viruses were generated by synthesis of cDNAs see Materials and Methods in plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo . Animals were immunized with each expression vector intramuscularly IM at three week intervals, and the antisera were evaluated on day 14 after the third immunization for their ability to neutralize HPAI H5N1 pseudotyped lentiviral vectors as previously described . We have previously shown that lentiviral assay inhibition LAI yields similar results to microneutralization and HAI analyses with higher sensitivity in mice To determine whether immunization with multiple HAs simultaneously could expand the breadth of the neutralizing antibody response without significant loss of magnitude, a combination of 10 HA DNA vaccine immunogens was administered IM at proportionally lower concentration 1.5 mg per immunogen into groups of 10 mice see Materials and Methods . Remarkably, despite a log lower DNA concentration of each component, significant neutralizing antibody titers were generated to each of the 10 immunogens, with .80% neutralization against 6 out of 12 H5 HA pseudoviruses at dilutions of up to 1:400 Fig. 2A . To evaluate whether similar breadth of immunity could be generated with fewer immunogens, two different combinations of 5 immunogens were selected, based on the phylogenetic diversity of HA among the avian influenza viruses and the crossreactivity of the neutralizing antibody responses of select individual immunogens Fig. 1 . As expected, there were substantial differences in the breadth of neutralization between these two sets of 5 immunogen multivalent vaccines Fig. 2 , B vs. C . In one set, while neutralization of homologous strains was comparable to the monovalent and the 10 immunogen multivalent immune response, fewer cross-reactive antibodies were detected, directed most prominently against A/Iraq Protection of DNA-vaccinated mice against challenge with heterologous H5N1 A/Vietnam/1203/2004 influenza virus Mice immunized as described above were challenged with a heterologous H5N1 virus 68 weeks after the final DNA vaccination. Animals were then challenged with 10 LD 50 of the highly pathogenic A/Vietnam/1203/2004 virus intranasally, and morbidity and mortality were monitored for 21 days after the viral challenge. The control animals, injected with the plasmid expression vector with no insert, died within 10 days of infection. Complete survival was observed in the groups immunized with the 10 component and set 2 of the 5 component multivalent DNA vaccines Fig. 3 . Immunization with HA derived from the A/ Indonesia/05/2005 strain or set 1 of the 5 component multivalent DNA vaccine showed a survival rate approaching 90%. In contrast, animals injected with HA plasmid DNA derived from A/ Anhui/1/2005, which has diverged more from A/Vietnam/ 1203/2004, showed a lower percent survival 70% after lethal viral challenge. Survival differences between groups were assessed using a log-rank test and the Gehan-Wilcoxon test on the survival curves for pairs of groups. A test was deemed significant if the pvalue was ,0.01. Mice injected IM with different HAs, A/ Indonesia/5/05, A/Anhui/1/05, 10HA, 5 HA Set 1 , or 5 HA Set 2 showed a significant difference compared to control all p values,0.001 . Among the HA-immunized groups, there was no significant difference between any two groups p.0.08 for all comparisons . For example, no significant difference was observed between the A/Anhui/1/05 group, which had the least survival among the HA immunized groups 7 out of 10 , and other HA groups: A/Indonesia/5/05 p = 0.377 , 10 HA p = 0.082 , 5 HA Set 1 p = 0.101 , or 5 HA Set 2 p = .411 . Therefore, we cannot exclude the possibility that the 3 deaths in the A/Anhui/1/05 group may have been due to random chance. Since it is desirable to confer protective immunity in poultry and HA DNA vaccination was effective in mice, we next examined the breadth and potency of single or multiple HA plasmid immunization in chickens. The ability of chickens to generate specific antibodies was assessed with three strains that showed broad cross protection in mouse studies A/Vietnam/1203/2004, A/Anhui/ 1/2005 and A/Indonesia/05/2005 , administered individually or in combination, by different injection methods. In addition to needle injection, a needle-free repetitive injection device, Agro-JetH Medical International Technology, Inc., Denver, CO , was analyzed. This device disperses the 0.1 to 5 ml injection doses into the dermal, subcutaneous, or intramuscular tissue depending upon the pressure adjustments, powered by a CO 2 gas pressure plunger . The injection conditions were determined by histologic analysis of tissues that received injections of India ink; a pressure of 48 psi was chosen since it enabled consistent delivery into intradermal and subcutaneous tissues Fig. S1 . Immunization of chickens with the control plasmid CMV/R without an HA gene insert elicited minimal neutralizing antibody titers compared to HA-immunized animals 1 week after 3 DNA immunizations. Nearly all chickens immunized with either monovalent or multivalent HA DNA vaccines generated significant neutralization titers Fig. 4 and Table S1 . In general, there was a progressive increase in the amount of neutralization after each successive DNA vaccination data not shown with maximal response at 1 week after the 3 rd DNA immunization, with highest and most consistent levels in the trivalent vaccine group delivered with the Agro-JetH device. Neutralization of the Indonesia HA strain was the most robust, with neutralization nearing 100% at titers greater than 1:3200. Both the monovalent and multivalent vaccines elicited robust homologous vaccine Fig. 4 . Even though one chicken . in the multivalent vaccine group produced almost the same degree of neutralization at each time point and was protected, it did not produce a high neutralizing antibody titer for reasons that were uncertain but possibly related to a non-specific inhibitor in the sera. To determine whether chickens immunized with single or multiple DNA vaccines were protected from a lethal challenge of a heterologous HPAI H5N1 virus, vaccinated chickens were In panels B and C, mice n = 10 were immunized with 15 mg of plasmid 3 mg each three times at 3 week intervals. Serum pools from the immunized animals were collected 14 days after the third immunization. The antisera were tested against the 12 indicated pseudotyped lentiviral vectors at varying dilutions. Error bars at each point indicate the standard deviation; each sample was evaluated in triplicate. In general, the immunized serum neutralized all tested pseudotyped lentiviruses at low dilutions while differences were often observed at high dilution. .1371/journal.pone.0002432.g002 inoculated with 20 LD 50 of highly pathogenic A/Vietnam/1203/ 2004 heterologous virus intranasally using standard methods and monitored for morbidity, mortality, viral shedding and serum antibodies. While all the control animals died within 2 days of infection, 100% survival was noted in the rest of the chickens Fig. 5A . The animals that were healthy, showing no signs of clinical disease or malaise, were euthanized on day 14. There was no evidence for viral shedding monitored via tracheal and cloacal swabs of infected chickens 2-14 days after challenge as determined by embryonal inoculation data not shown: egg infectious dose 50 EID 50 limit of detection ,100 virus particles . To compare the relative efficacy of DNA vaccines delivered IM by needle and syringe versus the needle-free Agro-JetH device injection, a dose-response study was performed with amounts of DNA vaccine ranging from 500 to 0.5 mg with two inoculations. In these experiments, the HA derived from A/chicken/Nigeria/641/ 2006 was substituted for A/Vietnam/1203/2004 since it represented a more contemporary isolate. The observed rate of protection was higher among the animals receiving 5 mg by Agro-Jet 8/8 than by IM injection 6/8 Fig. 5, B vs. C . Both modes provided complete protection for all animals at doses higher than this, and 25% protection for the animals receiving 0.5 mg doses Fig. 5B, C . Survival differences between consecutive doses were assessed using a log-rank test on the survival curves for pairs of groups. A test was deemed significant if the p-value was ,.01, and marginally significant if the p-value was ,.05 but ..01. Chickens injected IM showed a marginally significant difference between 0.5 and 5 mg p = .047 . In the same group there was a significant difference between control and 5, 50 and 500 mg p,.001 for all comparisons and the difference between control and 0.5 mg was marginally significant p = .016 . Chickens that were injected using Agro-JetH showed a significant difference between 0.5 and 5 mg p = .004 and between control and 5, 50, and 500 mg p,.001 for all comparisons . There were no differences between control and 0.5 mg or between 5, 50, and 500 mg. Lastly, the survival differences between Agro-JetH and IM for each dose group were not significant. The neutralizing antibody response to homologous and heterologous HAs corresponded with protection and correlated with dose, with higher titers elicited by injection with Agro-JetH compared to needle Table S2 . We assessed viable viral shedding after inoculation by chick embryo inoculation three days after virus challenge Week 8 . While we noted some embryonic lethality at the 0.5 mg dose, there was no embryonic lethality at 5, 50 or 500 mg groups data not shown . Since the HPAI H5N1 virus first appeared ten years ago, this highly pathogenic avian influenza virus has shown increasing diversification and dissemination in Asia, Africa, and Europe 28, . In addition to its effects on human health by crossspecies transmission and ability to compromise food sources, it poses a continuing threat to public health as it evolves and adapts in different species. The pandemic potential of this virus, especially as it relates to the poultry industry and for reservoir avian hosts, underscores the need for a vaccine that offers broad spectrum immunity and protection against lethal viral challenge. While the virus remains restricted in its ability to infect humans and undergo efficient human-to-human transmission , its persistence and spread in poultry increases the risk of the emergence of a pandemic strain. One approach to pandemic risk reduction is to limit the propagation of the virus in poultry and other relevant avian species. We have previously reported that DNA vaccines encoding HA can confer protection against a highly lethal human pandemic influenza virus, the 1918 H1N1 virus, in mice . DNA vaccines offer several advantages, including the ability to express diverse antigens, tolerability in various hosts, ease of delivery, and stability for storage and distribution without the necessity of maintaining a cold chain; they have been shown to be safe and efficacious in a variety of animal models . Because they do not contain other viral proteins used to screen for infection, they also address the need to differentiate vaccinated from infected animals. There is evidence that DNA vaccination elicits cell-mediated immunity against influenza HA in addition to inducing an antibody response , an effect that could significantly contribute to protective immunity as viruses show genetic drift and reduced susceptibility to neutralization. Ideally, a highly effective influenza vaccine should not only be able to let the host develop a protective immune response against a matching live virus challenge but also elicit robust protective immune responses against a broad range of homologous and heterologous H5 influenza strains. A multivalent H5 vaccine containing diverse serotypes could expand the antigenic breadth sufficiently to provide protection against heterologous challenge and may preclude the emergence of vaccine-resistant strains that may arise due to evolutionary vaccine pressure on the virus. Due to the antigenic drift and shift of the influenza virus genome, it has been very difficult to predict the next dominant strain of an avian endemic outbreak. DNA vaccines can be synthesized in a relatively short period of time, and the targeted mutations can be tailored to specific viral serotypes. The mutations promote a focused and enhanced immune response that may be particularly important in the event of an outbreak where specificity is the key to epidemic control. The use of modified codons ensures maximal expression in the host and eliminates the possibility of recombination with influenza viruses that might potentially generate new strains. A more broadly protective murine vaccine was developed here by including more HAs from varying strains in the multivalent vaccine Figs. 2 and 3 . However, it is less practical to include large numbers of different HAs in one vaccine due to the cost and complexity of manufacturing such a vaccine. Therefore, we simplified the vaccine regimen based on cross-neutralization studies and phylogenetic relationships. A trivalent vaccine was subsequently identified for further studies. Due . While three DNA immunizations were used initially to demonstrate protective immunity and have been used previously to elicit protection in mice , we found that effective protective immunity could be induced with two DNA vaccinations and as little as 5 mg trivalent DNA immunization using the ID/SC route with the Agro-JetH device. In addition, based on the chick embryo inoculation data, we believe that there is effective neutralization of the virus and lack of infectious viral shedding in chicken vaccinated with as little as 5 mg of DNA. The device's capacity for rapid repetitive injection and the lower quantity and stability of DNA enhance the practicality and utility of this approach for vaccination of endangered species in captivity or administration to poultry or other animals. A/Vietnam/1203/2004 H5N1 A/VN/1203/04 was obtained from the repository at the Centers for Disease Control and Prevention CDC , Atlanta, Georgia. The virus was propagated in 10-day old embryonated chicken eggs at 35uC and stored at 270uC until use. The virus was titrated by the Reed and Muench method to determine EID 50 . GenBank ABD28180 were synthesized using human-preferred codons GeneArt, Regensburg, Germany . HA cDNAs from diverse strains of influenza viruses were then inserted into plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo . For initial trivalent immunizations in chickens, the A/Vietnam/1203/ 2004, A/Anhui/1/2005 and A/Indonesia/05/2005 strains were used and in the dose response study, the Vietnam strain was replaced with A/chicken/Nigeria/641/2006. The immunogens used in DNA vaccination contained a cleavage site mutation PQRERRRKKRG to PQRETRG as previously described . This mutation was generated by site-directed mutagenesis using a QuickChange kit Stratagene, La Jolla, CA . DNA immunization of mice week old female BALB/c mice were purchased from The Jackson Laboratory and maintained in the AAALAC-accredited Vaccine Research Center Animal Care Facility Bethesda, MD under specific pathogen-free conditions. All experiments were approved by the Vaccine Research Center Animal Care and Use Committee. The mice were immunized as previously described . Briefly, mice 10 animals for all test groups, 20 animals for the The study was carried out in the AAALAC-accredited animal facility at the University of Maryland School of Medicine. Six groups of 8 one-day-old male and female SPAFAS White Leghorn Chickens, Gallus domesticus, were obtained from Charles River Laboratories Connecticut . The animals were housed in brooder and grower cages McMurray Hatcheries, Iowa . Feed Teklad Japanese Quail Diet -3050, Harlan-Teklad, WI and water were provided to the animals ad libitum. The study was performed in strict accordance with the ''Guide'' after approvals from the Animal Care and Use Committees of the Vaccine Research Center, NIH and the University of Maryland. DNA immunizations were performed as described at 0, 3 and 6 weeks. A total dose of 500 mg of one or a combination of the following DNA plasmids in a volume of 250 ml was administered to each animal: pCMV/ R, pCMV/R-HA Agro-JetH is a needle-free device used for mass delivery of vaccines and drugs in livestock and poultry. The device is semiautomatic and requires a small CO 2 tank or compressed air for low pressure delivery. Upon trigger activation, CO 2 disperses the injectate at a precise dose into the muscle, dermis or subcutaneous tissue depending on the setting that was standardized for our use. We used an effective volume of 0.1 ml in our injectate . In this study we were able to effectively deliver 0.1 ml of injectate into the animal's dermis/subcutaneous tissue at a pressure of 48-55 psi. Sixty-eight weeks after the last immunization, female BALB/c mice were lightly anesthetized with Ketamine/Xylazine and inoculated intranasally with 10 LD 50 of A/Vietnam/1203/2004 virus diluted in phosphate-buffered saline in a 50 ul volume. Mice were monitored daily for morbidity and measured for weight loss and mortality for 21 days post infection. Any mouse that had lost more than 25% of its body weight was euthanized. All experiments involving the HPAI virus were conducted in an AAALAC accredited facility BioQual Inc., Gaithersburg, MD under BSL 3 conditions that included enhancements required by the USDA and the Select Agent Program. White Leghorn chickens were challenged one week after the last immunization with 20 lethal dose 50 LD 50 of A/Vietnam/1203/04 H5N1 influenza A virus, equivalent to 2610 4 EID 50 based on previous challenges . Chickens were infected with 200 ml virus intranasally. Tracheal and cloacal swabs were collected days 3 and 5 post-challenge and stored in glass vials containing BHI medium BBL TM Brain Heart Infusion, Becton Dickinson at 280uC. Blood was collected 14 days post-challenge and serum was titered by microneutralization assay. Chickens were observed and scored daily for clinical signs of infection, morbidity and mortality. Chickens that survived the study were bled and humanely euthanized at day 14 post-challenge. Lungs, heart, intestine and kidney were collected and samples were stored in formalin for histopathology. Experiments were carried out under BSL3+ conditions with investigators wearing appropriate protective equipment and compliant with all Institutional Animal Care and Use Committee-approved protocols and under Animal Welfare Act regulations at the University of Maryland, College Park, Maryland. Representative tracheal and cloacal swabs were chosen to run an EID 50 assay for comparison and virus titers were determine by the method of Reed and Meunch . Briefly, swabs were used to infect 10 day-old embryonated chicken eggs in 10-fold dilutions. Three eggs were inoculated per dilution and incubated for 48 hours before titration. Neutralizing antibodies were titrated from serum samples collected week 5 and 7 post-vaccination and day 14 post-challenge. The microneutralization assay was performed using a 96-well plate format. Serum was treated with receptor-destroying enzyme Denka Seiken Co. and treated at 37uC per the manufacturer's instructions. After an overnight incubation and subsequent inactivation samples were brought to a final dilution of 1:10 using PBS and each sample was serially diluted and virus, diluted to 100 TCID 50 , was added to each well. The plates were then incubated at 37uC, 5% CO 2 for 1-2 hours. Following incubation, supernatants were used to infect a second 96-well plate of MDCK cells. Microplates were incubated at 4uC for 15 minutes and then 37uC, 5% CO 2 for 45 minutes. Supernatants of serum and virus were then discarded and 200 ml of OptiMEM containing 1X antibiotics/antimycotics, 1 mg/ml TPCK-trypsin was added and incubated at 37uC, 5% CO 2 for 3 days. After 3 days, 50 ml of the supernatant from each well was transferred into a new 96-well microplate, and an HA assay was performed to calculate the antibody titers. Virus and cell controls were included in the assay. Two-fold dilutions of heat-inactivated sera were tested in a microneutralization assay as previously described for the presence of antibodies that neutralized the infectivity of 100 TCID 50 50% tissue culture infectious dose of the A/Vietnam/ 1203/2004 H5N1 virus on MDCK cell monolayers by using two wells per dilution on a 96-well plate. The recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described . For the neutralization assay, antisera from immunized animals were heat-inactivated at 55uC for 30 minutes and mixed with 50 ml of pseudovirus at various dilutions. The sera/virus mixture was then added to 293A cells in 96-well B&W TC Isoplates Wallac, Turku, Finland; 12,000 cells/well . Two hours later, the plates were washed and fresh medium was added. Cells were lysed in mammalian cell lysis buffer Promega, Madison, WI 24 hrs after infection and luciferase activity was measured using the Luciferase Assay System Promega, Madison, WI . The following strains were used for the production of pseudotyped viruses: for HA we used A/Thailand/1 KAN- The HA/HI titers were determined as previously described . Briefly, HA titers were calculated using 50 ml of 0.5% chicken red blood cell suspension in PBS added to 50 ml of twofold dilutions of virus in PBS. This mix was incubated at room temperature for 30 minutes. The HA titers were calculated as the reciprocal value of the highest dilution that caused complete hemagglutination. HI titers were calculated by titrating 50 ml of antiserum treated with receptor-destroying enzyme and an equivalent amount of A/Vietnam/1203/2004 virus four hemagglutinating doses was added to each well. Wells were incubated at room temperature for 30 minutes and 50 ml of a 0.5% suspension of chicken red blood cells was added. HI titers were calculated after 30 minutes as the reciprocal of the serum dilution that inhibited hemagglutination. Table S1 Hemagglutination inhibition HI , microneutralization titer NT , and LAI of sera from individual chickens immunized with different vaccines. Sera from immunized animals were obtained at week 5 or 7, a week before or after the final boost, and neutralization was assessed by HI, microneutralization NT and LAI shown as IC 50 . Individual animal serum of each group is shown and was analyzed as described in the Materials and Methods section. Figure S1 Characterization of needle-free Agro-JetH DNA immunization in chickens. To evaluate the distribution of fluid into superficial or deep layers of subcutaneous tissues after delivery by AgroJetH, 4 or 7 week old chickens were injected with a solution containing India ink with this needle-free device at various pressures, ranging from 45 to 55 mm Hg. Three sites thigh, wing and breast were used, and biopsies were taken for routine hematoxylin and eosin staining. Representative sections of thigh injections are shown from 7 week old chickens and were similar at 4 weeks data not shown . While the 48 mm Hg pressure deposited the injectate into the dermis/subcutaneous region left , the higher pressure injections, 52 and 58 mm Hg, deposited the injectate into the subcutaneous and muscle layers middle, right . 48 mm Hg consistently provided an optimal pressure to deposit the injectate into the dermis and subcutaneous tissue and was chosen for all AgroJetH immunizations. Found at: .1371/journal.pone.0002432.s003 10.74 MB DOC
What is the conclusion of this study?	it is possible to develop a multivalent DNA vaccine for poultry that can protect against multiple HPAI H5N1 strains and that could keep pace with the continued evolution of avian influenza viruses	[ "BACKGROUND: Sustained outbreaks of highly pathogenic avian influenza HPAI H5N1 in avian species increase the risk of reassortment and adaptation to humans. The ability to contain its spread in chickens would reduce this threat and help maintain the capacity for egg-based vaccine production. While vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses. METHODOLOGY / PRINCIPAL FINDINGS: The ability of DNA vaccines encoding hemagglutinin HA proteins from different HPAI H5N1 serotypes was evaluated for its ability to elicit neutralizing antibodies and to protect against homologous and heterologous HPAI H5N1 strain challenge in mice and chickens after DNA immunization by needle and syringe or with a pressure injection device. These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs. The response was dose-dependent, and breadth was determined by the choice of the influenza virus HA in the vaccine.", "These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs. The response was dose-dependent, and breadth was determined by the choice of the influenza virus HA in the vaccine. Monovalent and trivalent HA vaccines were tested first in mice and conferred protection against lethal H5N1 A/Vietnam/1203/2004 challenge 68 weeks after vaccination. In chickens, protection was observed against heterologous strains of HPAI H5N1 after vaccination with a trivalent H5 serotype DNA vaccine with doses as low as 5 µg DNA given twice either by intramuscular needle injection or with a needle-free device. CONCLUSIONS/SIGNIFICANCE: DNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. In addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates.", "CONCLUSIONS/SIGNIFICANCE: DNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. In addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates. Text: The highly pathogenic H5N1 influenza virus causes lethal multi-organ disease in poultry, resulting in significant economic losses and a public health concern in many parts of the world. The greatest threats posed by this virus are its ability to cause mortality in humans, its potential to compromise food supplies, and its possible economic impacts. Viral maintenance in poultry potentiates the risk of human-to-human transmission and the emergence of a pandemic strain through reassortment. An effective, safe poultry vaccine that elicits broadly protective immune responses to evolving flu strains would provide a countermeasure to reduce the likelihood of transmission of this virus from domestic birds to humans and simultaneously would protect commercial poultry operations and subsistence farmers.", "Viral maintenance in poultry potentiates the risk of human-to-human transmission and the emergence of a pandemic strain through reassortment. An effective, safe poultry vaccine that elicits broadly protective immune responses to evolving flu strains would provide a countermeasure to reduce the likelihood of transmission of this virus from domestic birds to humans and simultaneously would protect commercial poultry operations and subsistence farmers. DNA vaccines have been shown to elicit robust immune responses in various animal species, from mice to nonhuman primates . In human trials, these vaccines elicit cellular and humoral immune responses against various infectious agents, including influenza, SARS, SIV and HIV. In addition to their ability to elicit antibody responses, they also stimulate antigenspecific and sustained T cell responses 6, 12, 13 . DNA vaccination has been used experimentally against various infectious agents in a variety of mammals, including cattle against infectious bovine rhinotracheitis/bovine diarrhea virus, leptospirosis and mycobacteriosis , pigs against classical swine fever virus and mycoplasmosis , and horses against West Nile virus and rabies .", "In addition to their ability to elicit antibody responses, they also stimulate antigenspecific and sustained T cell responses 6, 12, 13 . DNA vaccination has been used experimentally against various infectious agents in a variety of mammals, including cattle against infectious bovine rhinotracheitis/bovine diarrhea virus, leptospirosis and mycobacteriosis , pigs against classical swine fever virus and mycoplasmosis , and horses against West Nile virus and rabies . In addition, DNA vaccines have been tested against avian plasmodium infection in penguins and against influenza and infectious bursal disease in chickens , duck hepatitis B virus in ducks , and avian metapneumovirus and Chlamydia psittaci in turkeys reviewed in ref. . While they have been used in chickens to generate antisera to specific influenza viruses and confer protection against the low pathogenicity H5N2 strain , there is only one previous report of a monovalent DNA vaccine effective against H5N1 and that only against a matched H5N1 isolate ; no protection with multivalent DNA vaccines against heterologous strains has been reported. Development and characterization of a DNA vaccine modality for use in poultry offers a potential countermeasure against HPAI H5N1 avian influenza outbreaks.", "While they have been used in chickens to generate antisera to specific influenza viruses and confer protection against the low pathogenicity H5N2 strain , there is only one previous report of a monovalent DNA vaccine effective against H5N1 and that only against a matched H5N1 isolate ; no protection with multivalent DNA vaccines against heterologous strains has been reported. Development and characterization of a DNA vaccine modality for use in poultry offers a potential countermeasure against HPAI H5N1 avian influenza outbreaks. The virus can infect humans, typically from animal sources, including commercial and wild avian species, livestock, and possibly other non-domesticated animal species . While there is marked diversity in the host range of type A influenza viruses, many experts have speculated that a pandemic strain of type A influenza could evolve in avian species or avian influenza viruses could contribute virulent genes to a pandemic strain through reassortment . Thus, there is reason to consider vaccination of poultry that would stimulate potent and broad protective immune responses . In undertaking such efforts, it is important that there be a differentiation of infected from vaccinated animals so that animals can be protected and permit monitoring of new infections using proven and sensitive methodologies.", "Thus, there is reason to consider vaccination of poultry that would stimulate potent and broad protective immune responses . In undertaking such efforts, it is important that there be a differentiation of infected from vaccinated animals so that animals can be protected and permit monitoring of new infections using proven and sensitive methodologies. In this study, we used an automated high capacity needle-free injection device, Agro-JetH Medical International Technology, Inc., Denver, CO to explore the feasibility of DNA vaccination of poultry. After optimization of injection conditions, alternative multivalent DNA vaccine regimens were analyzed and compared for magnitude and breadth of neutralizing antibodies, as well as protective efficacy after challenge in mouse and chicken models of HPAI H5N1 infection. The findings suggest that it is possible to develop a multivalent DNA vaccine for poultry that can protect against multiple HPAI H5N1 strains and that could keep pace with the continued evolution of avian influenza viruses. Immunogenicity and neutralizing antibody specificity of alternative HA DNA vaccines in mice To evaluate the efficacy of multivalent DNA vaccines, initial studies were performed in mice.", "The findings suggest that it is possible to develop a multivalent DNA vaccine for poultry that can protect against multiple HPAI H5N1 strains and that could keep pace with the continued evolution of avian influenza viruses. Immunogenicity and neutralizing antibody specificity of alternative HA DNA vaccines in mice To evaluate the efficacy of multivalent DNA vaccines, initial studies were performed in mice. Expression vectors encoding HAs from ten phylogenetically diverse strains of influenza viruses were generated by synthesis of cDNAs see Materials and Methods in plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo . Animals were immunized with each expression vector intramuscularly IM at three week intervals, and the antisera were evaluated on day 14 after the third immunization for their ability to neutralize HPAI H5N1 pseudotyped lentiviral vectors as previously described . We have previously shown that lentiviral assay inhibition LAI yields similar results to microneutralization and HAI analyses with higher sensitivity in mice To determine whether immunization with multiple HAs simultaneously could expand the breadth of the neutralizing antibody response without significant loss of magnitude, a combination of 10 HA DNA vaccine immunogens was administered IM at proportionally lower concentration 1.5 mg per immunogen into groups of 10 mice see Materials and Methods . Remarkably, despite a log lower DNA concentration of each component, significant neutralizing antibody titers were generated to each of the 10 immunogens, with .80% neutralization against 6 out of 12 H5 HA pseudoviruses at dilutions of up to 1:400 Fig.", "We have previously shown that lentiviral assay inhibition LAI yields similar results to microneutralization and HAI analyses with higher sensitivity in mice To determine whether immunization with multiple HAs simultaneously could expand the breadth of the neutralizing antibody response without significant loss of magnitude, a combination of 10 HA DNA vaccine immunogens was administered IM at proportionally lower concentration 1.5 mg per immunogen into groups of 10 mice see Materials and Methods . Remarkably, despite a log lower DNA concentration of each component, significant neutralizing antibody titers were generated to each of the 10 immunogens, with .80% neutralization against 6 out of 12 H5 HA pseudoviruses at dilutions of up to 1:400 Fig. 2A . To evaluate whether similar breadth of immunity could be generated with fewer immunogens, two different combinations of 5 immunogens were selected, based on the phylogenetic diversity of HA among the avian influenza viruses and the crossreactivity of the neutralizing antibody responses of select individual immunogens Fig. 1 . As expected, there were substantial differences in the breadth of neutralization between these two sets of 5 immunogen multivalent vaccines Fig.", "1 . As expected, there were substantial differences in the breadth of neutralization between these two sets of 5 immunogen multivalent vaccines Fig. 2 , B vs. C . In one set, while neutralization of homologous strains was comparable to the monovalent and the 10 immunogen multivalent immune response, fewer cross-reactive antibodies were detected, directed most prominently against A/Iraq Protection of DNA-vaccinated mice against challenge with heterologous H5N1 A/Vietnam/1203/2004 influenza virus Mice immunized as described above were challenged with a heterologous H5N1 virus 68 weeks after the final DNA vaccination. Animals were then challenged with 10 LD 50 of the highly pathogenic A/Vietnam/1203/2004 virus intranasally, and morbidity and mortality were monitored for 21 days after the viral challenge. The control animals, injected with the plasmid expression vector with no insert, died within 10 days of infection.", "Animals were then challenged with 10 LD 50 of the highly pathogenic A/Vietnam/1203/2004 virus intranasally, and morbidity and mortality were monitored for 21 days after the viral challenge. The control animals, injected with the plasmid expression vector with no insert, died within 10 days of infection. Complete survival was observed in the groups immunized with the 10 component and set 2 of the 5 component multivalent DNA vaccines Fig. 3 . Immunization with HA derived from the A/ Indonesia/05/2005 strain or set 1 of the 5 component multivalent DNA vaccine showed a survival rate approaching 90%. In contrast, animals injected with HA plasmid DNA derived from A/ Anhui/1/2005, which has diverged more from A/Vietnam/ 1203/2004, showed a lower percent survival 70% after lethal viral challenge.", "Immunization with HA derived from the A/ Indonesia/05/2005 strain or set 1 of the 5 component multivalent DNA vaccine showed a survival rate approaching 90%. In contrast, animals injected with HA plasmid DNA derived from A/ Anhui/1/2005, which has diverged more from A/Vietnam/ 1203/2004, showed a lower percent survival 70% after lethal viral challenge. Survival differences between groups were assessed using a log-rank test and the Gehan-Wilcoxon test on the survival curves for pairs of groups. A test was deemed significant if the pvalue was ,0.01. Mice injected IM with different HAs, A/ Indonesia/5/05, A/Anhui/1/05, 10HA, 5 HA Set 1 , or 5 HA Set 2 showed a significant difference compared to control all p values,0.001 . Among the HA-immunized groups, there was no significant difference between any two groups p.0.08 for all comparisons .", "Mice injected IM with different HAs, A/ Indonesia/5/05, A/Anhui/1/05, 10HA, 5 HA Set 1 , or 5 HA Set 2 showed a significant difference compared to control all p values,0.001 . Among the HA-immunized groups, there was no significant difference between any two groups p.0.08 for all comparisons . For example, no significant difference was observed between the A/Anhui/1/05 group, which had the least survival among the HA immunized groups 7 out of 10 , and other HA groups: A/Indonesia/5/05 p = 0.377 , 10 HA p = 0.082 , 5 HA Set 1 p = 0.101 , or 5 HA Set 2 p = .411 . Therefore, we cannot exclude the possibility that the 3 deaths in the A/Anhui/1/05 group may have been due to random chance. Since it is desirable to confer protective immunity in poultry and HA DNA vaccination was effective in mice, we next examined the breadth and potency of single or multiple HA plasmid immunization in chickens. The ability of chickens to generate specific antibodies was assessed with three strains that showed broad cross protection in mouse studies A/Vietnam/1203/2004, A/Anhui/ 1/2005 and A/Indonesia/05/2005 , administered individually or in combination, by different injection methods.", "Since it is desirable to confer protective immunity in poultry and HA DNA vaccination was effective in mice, we next examined the breadth and potency of single or multiple HA plasmid immunization in chickens. The ability of chickens to generate specific antibodies was assessed with three strains that showed broad cross protection in mouse studies A/Vietnam/1203/2004, A/Anhui/ 1/2005 and A/Indonesia/05/2005 , administered individually or in combination, by different injection methods. In addition to needle injection, a needle-free repetitive injection device, Agro-JetH Medical International Technology, Inc., Denver, CO , was analyzed. This device disperses the 0.1 to 5 ml injection doses into the dermal, subcutaneous, or intramuscular tissue depending upon the pressure adjustments, powered by a CO 2 gas pressure plunger . The injection conditions were determined by histologic analysis of tissues that received injections of India ink; a pressure of 48 psi was chosen since it enabled consistent delivery into intradermal and subcutaneous tissues Fig. S1 .", "The injection conditions were determined by histologic analysis of tissues that received injections of India ink; a pressure of 48 psi was chosen since it enabled consistent delivery into intradermal and subcutaneous tissues Fig. S1 . Immunization of chickens with the control plasmid CMV/R without an HA gene insert elicited minimal neutralizing antibody titers compared to HA-immunized animals 1 week after 3 DNA immunizations. Nearly all chickens immunized with either monovalent or multivalent HA DNA vaccines generated significant neutralization titers Fig. 4 and Table S1 . In general, there was a progressive increase in the amount of neutralization after each successive DNA vaccination data not shown with maximal response at 1 week after the 3 rd DNA immunization, with highest and most consistent levels in the trivalent vaccine group delivered with the Agro-JetH device.", "4 and Table S1 . In general, there was a progressive increase in the amount of neutralization after each successive DNA vaccination data not shown with maximal response at 1 week after the 3 rd DNA immunization, with highest and most consistent levels in the trivalent vaccine group delivered with the Agro-JetH device. Neutralization of the Indonesia HA strain was the most robust, with neutralization nearing 100% at titers greater than 1:3200. Both the monovalent and multivalent vaccines elicited robust homologous vaccine Fig. 4 . Even though one chicken .", "Both the monovalent and multivalent vaccines elicited robust homologous vaccine Fig. 4 . Even though one chicken . in the multivalent vaccine group produced almost the same degree of neutralization at each time point and was protected, it did not produce a high neutralizing antibody titer for reasons that were uncertain but possibly related to a non-specific inhibitor in the sera. To determine whether chickens immunized with single or multiple DNA vaccines were protected from a lethal challenge of a heterologous HPAI H5N1 virus, vaccinated chickens were In panels B and C, mice n = 10 were immunized with 15 mg of plasmid 3 mg each three times at 3 week intervals. Serum pools from the immunized animals were collected 14 days after the third immunization.", "To determine whether chickens immunized with single or multiple DNA vaccines were protected from a lethal challenge of a heterologous HPAI H5N1 virus, vaccinated chickens were In panels B and C, mice n = 10 were immunized with 15 mg of plasmid 3 mg each three times at 3 week intervals. Serum pools from the immunized animals were collected 14 days after the third immunization. The antisera were tested against the 12 indicated pseudotyped lentiviral vectors at varying dilutions. Error bars at each point indicate the standard deviation; each sample was evaluated in triplicate. In general, the immunized serum neutralized all tested pseudotyped lentiviruses at low dilutions while differences were often observed at high dilution. .1371/journal.pone.0002432.g002 inoculated with 20 LD 50 of highly pathogenic A/Vietnam/1203/ 2004 heterologous virus intranasally using standard methods and monitored for morbidity, mortality, viral shedding and serum antibodies.", "In general, the immunized serum neutralized all tested pseudotyped lentiviruses at low dilutions while differences were often observed at high dilution. .1371/journal.pone.0002432.g002 inoculated with 20 LD 50 of highly pathogenic A/Vietnam/1203/ 2004 heterologous virus intranasally using standard methods and monitored for morbidity, mortality, viral shedding and serum antibodies. While all the control animals died within 2 days of infection, 100% survival was noted in the rest of the chickens Fig. 5A . The animals that were healthy, showing no signs of clinical disease or malaise, were euthanized on day 14. There was no evidence for viral shedding monitored via tracheal and cloacal swabs of infected chickens 2-14 days after challenge as determined by embryonal inoculation data not shown: egg infectious dose 50 EID 50 limit of detection ,100 virus particles .", "The animals that were healthy, showing no signs of clinical disease or malaise, were euthanized on day 14. There was no evidence for viral shedding monitored via tracheal and cloacal swabs of infected chickens 2-14 days after challenge as determined by embryonal inoculation data not shown: egg infectious dose 50 EID 50 limit of detection ,100 virus particles . To compare the relative efficacy of DNA vaccines delivered IM by needle and syringe versus the needle-free Agro-JetH device injection, a dose-response study was performed with amounts of DNA vaccine ranging from 500 to 0.5 mg with two inoculations. In these experiments, the HA derived from A/chicken/Nigeria/641/ 2006 was substituted for A/Vietnam/1203/2004 since it represented a more contemporary isolate. The observed rate of protection was higher among the animals receiving 5 mg by Agro-Jet 8/8 than by IM injection 6/8 Fig. 5, B vs. C .", "The observed rate of protection was higher among the animals receiving 5 mg by Agro-Jet 8/8 than by IM injection 6/8 Fig. 5, B vs. C . Both modes provided complete protection for all animals at doses higher than this, and 25% protection for the animals receiving 0.5 mg doses Fig. 5B, C . Survival differences between consecutive doses were assessed using a log-rank test on the survival curves for pairs of groups. A test was deemed significant if the p-value was ,.01, and marginally significant if the p-value was ,.05 but ..01. Chickens injected IM showed a marginally significant difference between 0.5 and 5 mg p = .047 .", "A test was deemed significant if the p-value was ,.01, and marginally significant if the p-value was ,.05 but ..01. Chickens injected IM showed a marginally significant difference between 0.5 and 5 mg p = .047 . In the same group there was a significant difference between control and 5, 50 and 500 mg p,.001 for all comparisons and the difference between control and 0.5 mg was marginally significant p = .016 . Chickens that were injected using Agro-JetH showed a significant difference between 0.5 and 5 mg p = .004 and between control and 5, 50, and 500 mg p,.001 for all comparisons . There were no differences between control and 0.5 mg or between 5, 50, and 500 mg. Lastly, the survival differences between Agro-JetH and IM for each dose group were not significant. The neutralizing antibody response to homologous and heterologous HAs corresponded with protection and correlated with dose, with higher titers elicited by injection with Agro-JetH compared to needle Table S2 .", "There were no differences between control and 0.5 mg or between 5, 50, and 500 mg. Lastly, the survival differences between Agro-JetH and IM for each dose group were not significant. The neutralizing antibody response to homologous and heterologous HAs corresponded with protection and correlated with dose, with higher titers elicited by injection with Agro-JetH compared to needle Table S2 . We assessed viable viral shedding after inoculation by chick embryo inoculation three days after virus challenge Week 8 . While we noted some embryonic lethality at the 0.5 mg dose, there was no embryonic lethality at 5, 50 or 500 mg groups data not shown . Since the HPAI H5N1 virus first appeared ten years ago, this highly pathogenic avian influenza virus has shown increasing diversification and dissemination in Asia, Africa, and Europe 28, . In addition to its effects on human health by crossspecies transmission and ability to compromise food sources, it poses a continuing threat to public health as it evolves and adapts in different species.", "Since the HPAI H5N1 virus first appeared ten years ago, this highly pathogenic avian influenza virus has shown increasing diversification and dissemination in Asia, Africa, and Europe 28, . In addition to its effects on human health by crossspecies transmission and ability to compromise food sources, it poses a continuing threat to public health as it evolves and adapts in different species. The pandemic potential of this virus, especially as it relates to the poultry industry and for reservoir avian hosts, underscores the need for a vaccine that offers broad spectrum immunity and protection against lethal viral challenge. While the virus remains restricted in its ability to infect humans and undergo efficient human-to-human transmission , its persistence and spread in poultry increases the risk of the emergence of a pandemic strain. One approach to pandemic risk reduction is to limit the propagation of the virus in poultry and other relevant avian species. We have previously reported that DNA vaccines encoding HA can confer protection against a highly lethal human pandemic influenza virus, the 1918 H1N1 virus, in mice .", "One approach to pandemic risk reduction is to limit the propagation of the virus in poultry and other relevant avian species. We have previously reported that DNA vaccines encoding HA can confer protection against a highly lethal human pandemic influenza virus, the 1918 H1N1 virus, in mice . DNA vaccines offer several advantages, including the ability to express diverse antigens, tolerability in various hosts, ease of delivery, and stability for storage and distribution without the necessity of maintaining a cold chain; they have been shown to be safe and efficacious in a variety of animal models . Because they do not contain other viral proteins used to screen for infection, they also address the need to differentiate vaccinated from infected animals. There is evidence that DNA vaccination elicits cell-mediated immunity against influenza HA in addition to inducing an antibody response , an effect that could significantly contribute to protective immunity as viruses show genetic drift and reduced susceptibility to neutralization. Ideally, a highly effective influenza vaccine should not only be able to let the host develop a protective immune response against a matching live virus challenge but also elicit robust protective immune responses against a broad range of homologous and heterologous H5 influenza strains.", "There is evidence that DNA vaccination elicits cell-mediated immunity against influenza HA in addition to inducing an antibody response , an effect that could significantly contribute to protective immunity as viruses show genetic drift and reduced susceptibility to neutralization. Ideally, a highly effective influenza vaccine should not only be able to let the host develop a protective immune response against a matching live virus challenge but also elicit robust protective immune responses against a broad range of homologous and heterologous H5 influenza strains. A multivalent H5 vaccine containing diverse serotypes could expand the antigenic breadth sufficiently to provide protection against heterologous challenge and may preclude the emergence of vaccine-resistant strains that may arise due to evolutionary vaccine pressure on the virus. Due to the antigenic drift and shift of the influenza virus genome, it has been very difficult to predict the next dominant strain of an avian endemic outbreak. DNA vaccines can be synthesized in a relatively short period of time, and the targeted mutations can be tailored to specific viral serotypes. The mutations promote a focused and enhanced immune response that may be particularly important in the event of an outbreak where specificity is the key to epidemic control.", "DNA vaccines can be synthesized in a relatively short period of time, and the targeted mutations can be tailored to specific viral serotypes. The mutations promote a focused and enhanced immune response that may be particularly important in the event of an outbreak where specificity is the key to epidemic control. The use of modified codons ensures maximal expression in the host and eliminates the possibility of recombination with influenza viruses that might potentially generate new strains. A more broadly protective murine vaccine was developed here by including more HAs from varying strains in the multivalent vaccine Figs. 2 and 3 . However, it is less practical to include large numbers of different HAs in one vaccine due to the cost and complexity of manufacturing such a vaccine.", "2 and 3 . However, it is less practical to include large numbers of different HAs in one vaccine due to the cost and complexity of manufacturing such a vaccine. Therefore, we simplified the vaccine regimen based on cross-neutralization studies and phylogenetic relationships. A trivalent vaccine was subsequently identified for further studies. Due . While three DNA immunizations were used initially to demonstrate protective immunity and have been used previously to elicit protection in mice , we found that effective protective immunity could be induced with two DNA vaccinations and as little as 5 mg trivalent DNA immunization using the ID/SC route with the Agro-JetH device.", "Due . While three DNA immunizations were used initially to demonstrate protective immunity and have been used previously to elicit protection in mice , we found that effective protective immunity could be induced with two DNA vaccinations and as little as 5 mg trivalent DNA immunization using the ID/SC route with the Agro-JetH device. In addition, based on the chick embryo inoculation data, we believe that there is effective neutralization of the virus and lack of infectious viral shedding in chicken vaccinated with as little as 5 mg of DNA. The device's capacity for rapid repetitive injection and the lower quantity and stability of DNA enhance the practicality and utility of this approach for vaccination of endangered species in captivity or administration to poultry or other animals. A/Vietnam/1203/2004 H5N1 A/VN/1203/04 was obtained from the repository at the Centers for Disease Control and Prevention CDC , Atlanta, Georgia. The virus was propagated in 10-day old embryonated chicken eggs at 35uC and stored at 270uC until use.", "A/Vietnam/1203/2004 H5N1 A/VN/1203/04 was obtained from the repository at the Centers for Disease Control and Prevention CDC , Atlanta, Georgia. The virus was propagated in 10-day old embryonated chicken eggs at 35uC and stored at 270uC until use. The virus was titrated by the Reed and Muench method to determine EID 50 . GenBank ABD28180 were synthesized using human-preferred codons GeneArt, Regensburg, Germany . HA cDNAs from diverse strains of influenza viruses were then inserted into plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo . For initial trivalent immunizations in chickens, the A/Vietnam/1203/ 2004, A/Anhui/1/2005 and A/Indonesia/05/2005 strains were used and in the dose response study, the Vietnam strain was replaced with A/chicken/Nigeria/641/2006.", "HA cDNAs from diverse strains of influenza viruses were then inserted into plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo . For initial trivalent immunizations in chickens, the A/Vietnam/1203/ 2004, A/Anhui/1/2005 and A/Indonesia/05/2005 strains were used and in the dose response study, the Vietnam strain was replaced with A/chicken/Nigeria/641/2006. The immunogens used in DNA vaccination contained a cleavage site mutation PQRERRRKKRG to PQRETRG as previously described . This mutation was generated by site-directed mutagenesis using a QuickChange kit Stratagene, La Jolla, CA . DNA immunization of mice week old female BALB/c mice were purchased from The Jackson Laboratory and maintained in the AAALAC-accredited Vaccine Research Center Animal Care Facility Bethesda, MD under specific pathogen-free conditions. All experiments were approved by the Vaccine Research Center Animal Care and Use Committee.", "DNA immunization of mice week old female BALB/c mice were purchased from The Jackson Laboratory and maintained in the AAALAC-accredited Vaccine Research Center Animal Care Facility Bethesda, MD under specific pathogen-free conditions. All experiments were approved by the Vaccine Research Center Animal Care and Use Committee. The mice were immunized as previously described . Briefly, mice 10 animals for all test groups, 20 animals for the The study was carried out in the AAALAC-accredited animal facility at the University of Maryland School of Medicine. Six groups of 8 one-day-old male and female SPAFAS White Leghorn Chickens, Gallus domesticus, were obtained from Charles River Laboratories Connecticut . The animals were housed in brooder and grower cages McMurray Hatcheries, Iowa .", "Six groups of 8 one-day-old male and female SPAFAS White Leghorn Chickens, Gallus domesticus, were obtained from Charles River Laboratories Connecticut . The animals were housed in brooder and grower cages McMurray Hatcheries, Iowa . Feed Teklad Japanese Quail Diet -3050, Harlan-Teklad, WI and water were provided to the animals ad libitum. The study was performed in strict accordance with the ''Guide'' after approvals from the Animal Care and Use Committees of the Vaccine Research Center, NIH and the University of Maryland. DNA immunizations were performed as described at 0, 3 and 6 weeks. A total dose of 500 mg of one or a combination of the following DNA plasmids in a volume of 250 ml was administered to each animal: pCMV/ R, pCMV/R-HA Agro-JetH is a needle-free device used for mass delivery of vaccines and drugs in livestock and poultry.", "DNA immunizations were performed as described at 0, 3 and 6 weeks. A total dose of 500 mg of one or a combination of the following DNA plasmids in a volume of 250 ml was administered to each animal: pCMV/ R, pCMV/R-HA Agro-JetH is a needle-free device used for mass delivery of vaccines and drugs in livestock and poultry. The device is semiautomatic and requires a small CO 2 tank or compressed air for low pressure delivery. Upon trigger activation, CO 2 disperses the injectate at a precise dose into the muscle, dermis or subcutaneous tissue depending on the setting that was standardized for our use. We used an effective volume of 0.1 ml in our injectate . In this study we were able to effectively deliver 0.1 ml of injectate into the animal's dermis/subcutaneous tissue at a pressure of 48-55 psi.", "We used an effective volume of 0.1 ml in our injectate . In this study we were able to effectively deliver 0.1 ml of injectate into the animal's dermis/subcutaneous tissue at a pressure of 48-55 psi. Sixty-eight weeks after the last immunization, female BALB/c mice were lightly anesthetized with Ketamine/Xylazine and inoculated intranasally with 10 LD 50 of A/Vietnam/1203/2004 virus diluted in phosphate-buffered saline in a 50 ul volume. Mice were monitored daily for morbidity and measured for weight loss and mortality for 21 days post infection. Any mouse that had lost more than 25% of its body weight was euthanized. All experiments involving the HPAI virus were conducted in an AAALAC accredited facility BioQual Inc., Gaithersburg, MD under BSL 3 conditions that included enhancements required by the USDA and the Select Agent Program.", "Any mouse that had lost more than 25% of its body weight was euthanized. All experiments involving the HPAI virus were conducted in an AAALAC accredited facility BioQual Inc., Gaithersburg, MD under BSL 3 conditions that included enhancements required by the USDA and the Select Agent Program. White Leghorn chickens were challenged one week after the last immunization with 20 lethal dose 50 LD 50 of A/Vietnam/1203/04 H5N1 influenza A virus, equivalent to 2610 4 EID 50 based on previous challenges . Chickens were infected with 200 ml virus intranasally. Tracheal and cloacal swabs were collected days 3 and 5 post-challenge and stored in glass vials containing BHI medium BBL TM Brain Heart Infusion, Becton Dickinson at 280uC. Blood was collected 14 days post-challenge and serum was titered by microneutralization assay.", "Tracheal and cloacal swabs were collected days 3 and 5 post-challenge and stored in glass vials containing BHI medium BBL TM Brain Heart Infusion, Becton Dickinson at 280uC. Blood was collected 14 days post-challenge and serum was titered by microneutralization assay. Chickens were observed and scored daily for clinical signs of infection, morbidity and mortality. Chickens that survived the study were bled and humanely euthanized at day 14 post-challenge. Lungs, heart, intestine and kidney were collected and samples were stored in formalin for histopathology. Experiments were carried out under BSL3+ conditions with investigators wearing appropriate protective equipment and compliant with all Institutional Animal Care and Use Committee-approved protocols and under Animal Welfare Act regulations at the University of Maryland, College Park, Maryland.", "Lungs, heart, intestine and kidney were collected and samples were stored in formalin for histopathology. Experiments were carried out under BSL3+ conditions with investigators wearing appropriate protective equipment and compliant with all Institutional Animal Care and Use Committee-approved protocols and under Animal Welfare Act regulations at the University of Maryland, College Park, Maryland. Representative tracheal and cloacal swabs were chosen to run an EID 50 assay for comparison and virus titers were determine by the method of Reed and Meunch . Briefly, swabs were used to infect 10 day-old embryonated chicken eggs in 10-fold dilutions. Three eggs were inoculated per dilution and incubated for 48 hours before titration. Neutralizing antibodies were titrated from serum samples collected week 5 and 7 post-vaccination and day 14 post-challenge.", "Three eggs were inoculated per dilution and incubated for 48 hours before titration. Neutralizing antibodies were titrated from serum samples collected week 5 and 7 post-vaccination and day 14 post-challenge. The microneutralization assay was performed using a 96-well plate format. Serum was treated with receptor-destroying enzyme Denka Seiken Co. and treated at 37uC per the manufacturer's instructions. After an overnight incubation and subsequent inactivation samples were brought to a final dilution of 1:10 using PBS and each sample was serially diluted and virus, diluted to 100 TCID 50 , was added to each well. The plates were then incubated at 37uC, 5% CO 2 for 1-2 hours.", "After an overnight incubation and subsequent inactivation samples were brought to a final dilution of 1:10 using PBS and each sample was serially diluted and virus, diluted to 100 TCID 50 , was added to each well. The plates were then incubated at 37uC, 5% CO 2 for 1-2 hours. Following incubation, supernatants were used to infect a second 96-well plate of MDCK cells. Microplates were incubated at 4uC for 15 minutes and then 37uC, 5% CO 2 for 45 minutes. Supernatants of serum and virus were then discarded and 200 ml of OptiMEM containing 1X antibiotics/antimycotics, 1 mg/ml TPCK-trypsin was added and incubated at 37uC, 5% CO 2 for 3 days. After 3 days, 50 ml of the supernatant from each well was transferred into a new 96-well microplate, and an HA assay was performed to calculate the antibody titers.", "Supernatants of serum and virus were then discarded and 200 ml of OptiMEM containing 1X antibiotics/antimycotics, 1 mg/ml TPCK-trypsin was added and incubated at 37uC, 5% CO 2 for 3 days. After 3 days, 50 ml of the supernatant from each well was transferred into a new 96-well microplate, and an HA assay was performed to calculate the antibody titers. Virus and cell controls were included in the assay. Two-fold dilutions of heat-inactivated sera were tested in a microneutralization assay as previously described for the presence of antibodies that neutralized the infectivity of 100 TCID 50 50% tissue culture infectious dose of the A/Vietnam/ 1203/2004 H5N1 virus on MDCK cell monolayers by using two wells per dilution on a 96-well plate. The recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described . For the neutralization assay, antisera from immunized animals were heat-inactivated at 55uC for 30 minutes and mixed with 50 ml of pseudovirus at various dilutions.", "The recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described . For the neutralization assay, antisera from immunized animals were heat-inactivated at 55uC for 30 minutes and mixed with 50 ml of pseudovirus at various dilutions. The sera/virus mixture was then added to 293A cells in 96-well B&W TC Isoplates Wallac, Turku, Finland; 12,000 cells/well . Two hours later, the plates were washed and fresh medium was added. Cells were lysed in mammalian cell lysis buffer Promega, Madison, WI 24 hrs after infection and luciferase activity was measured using the Luciferase Assay System Promega, Madison, WI . The following strains were used for the production of pseudotyped viruses: for HA we used A/Thailand/1 KAN- The HA/HI titers were determined as previously described .", "Cells were lysed in mammalian cell lysis buffer Promega, Madison, WI 24 hrs after infection and luciferase activity was measured using the Luciferase Assay System Promega, Madison, WI . The following strains were used for the production of pseudotyped viruses: for HA we used A/Thailand/1 KAN- The HA/HI titers were determined as previously described . Briefly, HA titers were calculated using 50 ml of 0.5% chicken red blood cell suspension in PBS added to 50 ml of twofold dilutions of virus in PBS. This mix was incubated at room temperature for 30 minutes. The HA titers were calculated as the reciprocal value of the highest dilution that caused complete hemagglutination. HI titers were calculated by titrating 50 ml of antiserum treated with receptor-destroying enzyme and an equivalent amount of A/Vietnam/1203/2004 virus four hemagglutinating doses was added to each well.", "The HA titers were calculated as the reciprocal value of the highest dilution that caused complete hemagglutination. HI titers were calculated by titrating 50 ml of antiserum treated with receptor-destroying enzyme and an equivalent amount of A/Vietnam/1203/2004 virus four hemagglutinating doses was added to each well. Wells were incubated at room temperature for 30 minutes and 50 ml of a 0.5% suspension of chicken red blood cells was added. HI titers were calculated after 30 minutes as the reciprocal of the serum dilution that inhibited hemagglutination. Table S1 Hemagglutination inhibition HI , microneutralization titer NT , and LAI of sera from individual chickens immunized with different vaccines. Sera from immunized animals were obtained at week 5 or 7, a week before or after the final boost, and neutralization was assessed by HI, microneutralization NT and LAI shown as IC 50 .", "Table S1 Hemagglutination inhibition HI , microneutralization titer NT , and LAI of sera from individual chickens immunized with different vaccines. Sera from immunized animals were obtained at week 5 or 7, a week before or after the final boost, and neutralization was assessed by HI, microneutralization NT and LAI shown as IC 50 . Individual animal serum of each group is shown and was analyzed as described in the Materials and Methods section. Figure S1 Characterization of needle-free Agro-JetH DNA immunization in chickens. To evaluate the distribution of fluid into superficial or deep layers of subcutaneous tissues after delivery by AgroJetH, 4 or 7 week old chickens were injected with a solution containing India ink with this needle-free device at various pressures, ranging from 45 to 55 mm Hg. Three sites thigh, wing and breast were used, and biopsies were taken for routine hematoxylin and eosin staining.", "To evaluate the distribution of fluid into superficial or deep layers of subcutaneous tissues after delivery by AgroJetH, 4 or 7 week old chickens were injected with a solution containing India ink with this needle-free device at various pressures, ranging from 45 to 55 mm Hg. Three sites thigh, wing and breast were used, and biopsies were taken for routine hematoxylin and eosin staining. Representative sections of thigh injections are shown from 7 week old chickens and were similar at 4 weeks data not shown . While the 48 mm Hg pressure deposited the injectate into the dermis/subcutaneous region left , the higher pressure injections, 52 and 58 mm Hg, deposited the injectate into the subcutaneous and muscle layers middle, right . 48 mm Hg consistently provided an optimal pressure to deposit the injectate into the dermis and subcutaneous tissue and was chosen for all AgroJetH immunizations. Found at: .1371/journal.pone.0002432.s003 10.74 MB DOC" ]	1,608	5,286	BACKGROUND: Sustained outbreaks of highly pathogenic avian influenza HPAI H5N1 in avian species increase the risk of reassortment and adaptation to humans. The ability to contain its spread in chickens would reduce this threat and help maintain the capacity for egg-based vaccine production. While vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses. METHODOLOGY / PRINCIPAL FINDINGS: The ability of DNA vaccines encoding hemagglutinin HA proteins from different HPAI H5N1 serotypes was evaluated for its ability to elicit neutralizing antibodies and to protect against homologous and heterologous HPAI H5N1 strain challenge in mice and chickens after DNA immunization by needle and syringe or with a pressure injection device. These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs. The response was dose-dependent, and breadth was determined by the choice of the influenza virus HA in the vaccine. Monovalent and trivalent HA vaccines were tested first in mice and conferred protection against lethal H5N1 A/Vietnam/1203/2004 challenge 68 weeks after vaccination. In chickens, protection was observed against heterologous strains of HPAI H5N1 after vaccination with a trivalent H5 serotype DNA vaccine with doses as low as 5 µg DNA given twice either by intramuscular needle injection or with a needle-free device. CONCLUSIONS/SIGNIFICANCE: DNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. In addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates. Text: The highly pathogenic H5N1 influenza virus causes lethal multi-organ disease in poultry, resulting in significant economic losses and a public health concern in many parts of the world. The greatest threats posed by this virus are its ability to cause mortality in humans, its potential to compromise food supplies, and its possible economic impacts. Viral maintenance in poultry potentiates the risk of human-to-human transmission and the emergence of a pandemic strain through reassortment. An effective, safe poultry vaccine that elicits broadly protective immune responses to evolving flu strains would provide a countermeasure to reduce the likelihood of transmission of this virus from domestic birds to humans and simultaneously would protect commercial poultry operations and subsistence farmers. DNA vaccines have been shown to elicit robust immune responses in various animal species, from mice to nonhuman primates . In human trials, these vaccines elicit cellular and humoral immune responses against various infectious agents, including influenza, SARS, SIV and HIV. In addition to their ability to elicit antibody responses, they also stimulate antigenspecific and sustained T cell responses 6, 12, 13 . DNA vaccination has been used experimentally against various infectious agents in a variety of mammals, including cattle against infectious bovine rhinotracheitis/bovine diarrhea virus, leptospirosis and mycobacteriosis , pigs against classical swine fever virus and mycoplasmosis , and horses against West Nile virus and rabies . In addition, DNA vaccines have been tested against avian plasmodium infection in penguins and against influenza and infectious bursal disease in chickens , duck hepatitis B virus in ducks , and avian metapneumovirus and Chlamydia psittaci in turkeys reviewed in ref. . While they have been used in chickens to generate antisera to specific influenza viruses and confer protection against the low pathogenicity H5N2 strain , there is only one previous report of a monovalent DNA vaccine effective against H5N1 and that only against a matched H5N1 isolate ; no protection with multivalent DNA vaccines against heterologous strains has been reported. Development and characterization of a DNA vaccine modality for use in poultry offers a potential countermeasure against HPAI H5N1 avian influenza outbreaks. The virus can infect humans, typically from animal sources, including commercial and wild avian species, livestock, and possibly other non-domesticated animal species . While there is marked diversity in the host range of type A influenza viruses, many experts have speculated that a pandemic strain of type A influenza could evolve in avian species or avian influenza viruses could contribute virulent genes to a pandemic strain through reassortment . Thus, there is reason to consider vaccination of poultry that would stimulate potent and broad protective immune responses . In undertaking such efforts, it is important that there be a differentiation of infected from vaccinated animals so that animals can be protected and permit monitoring of new infections using proven and sensitive methodologies. In this study, we used an automated high capacity needle-free injection device, Agro-JetH Medical International Technology, Inc., Denver, CO to explore the feasibility of DNA vaccination of poultry. After optimization of injection conditions, alternative multivalent DNA vaccine regimens were analyzed and compared for magnitude and breadth of neutralizing antibodies, as well as protective efficacy after challenge in mouse and chicken models of HPAI H5N1 infection. The findings suggest that it is possible to develop a multivalent DNA vaccine for poultry that can protect against multiple HPAI H5N1 strains and that could keep pace with the continued evolution of avian influenza viruses. Immunogenicity and neutralizing antibody specificity of alternative HA DNA vaccines in mice To evaluate the efficacy of multivalent DNA vaccines, initial studies were performed in mice. Expression vectors encoding HAs from ten phylogenetically diverse strains of influenza viruses were generated by synthesis of cDNAs see Materials and Methods in plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo . Animals were immunized with each expression vector intramuscularly IM at three week intervals, and the antisera were evaluated on day 14 after the third immunization for their ability to neutralize HPAI H5N1 pseudotyped lentiviral vectors as previously described . We have previously shown that lentiviral assay inhibition LAI yields similar results to microneutralization and HAI analyses with higher sensitivity in mice To determine whether immunization with multiple HAs simultaneously could expand the breadth of the neutralizing antibody response without significant loss of magnitude, a combination of 10 HA DNA vaccine immunogens was administered IM at proportionally lower concentration 1.5 mg per immunogen into groups of 10 mice see Materials and Methods . Remarkably, despite a log lower DNA concentration of each component, significant neutralizing antibody titers were generated to each of the 10 immunogens, with .80% neutralization against 6 out of 12 H5 HA pseudoviruses at dilutions of up to 1:400 Fig. 2A . To evaluate whether similar breadth of immunity could be generated with fewer immunogens, two different combinations of 5 immunogens were selected, based on the phylogenetic diversity of HA among the avian influenza viruses and the crossreactivity of the neutralizing antibody responses of select individual immunogens Fig. 1 . As expected, there were substantial differences in the breadth of neutralization between these two sets of 5 immunogen multivalent vaccines Fig. 2 , B vs. C . In one set, while neutralization of homologous strains was comparable to the monovalent and the 10 immunogen multivalent immune response, fewer cross-reactive antibodies were detected, directed most prominently against A/Iraq Protection of DNA-vaccinated mice against challenge with heterologous H5N1 A/Vietnam/1203/2004 influenza virus Mice immunized as described above were challenged with a heterologous H5N1 virus 68 weeks after the final DNA vaccination. Animals were then challenged with 10 LD 50 of the highly pathogenic A/Vietnam/1203/2004 virus intranasally, and morbidity and mortality were monitored for 21 days after the viral challenge. The control animals, injected with the plasmid expression vector with no insert, died within 10 days of infection. Complete survival was observed in the groups immunized with the 10 component and set 2 of the 5 component multivalent DNA vaccines Fig. 3 . Immunization with HA derived from the A/ Indonesia/05/2005 strain or set 1 of the 5 component multivalent DNA vaccine showed a survival rate approaching 90%. In contrast, animals injected with HA plasmid DNA derived from A/ Anhui/1/2005, which has diverged more from A/Vietnam/ 1203/2004, showed a lower percent survival 70% after lethal viral challenge. Survival differences between groups were assessed using a log-rank test and the Gehan-Wilcoxon test on the survival curves for pairs of groups. A test was deemed significant if the pvalue was ,0.01. Mice injected IM with different HAs, A/ Indonesia/5/05, A/Anhui/1/05, 10HA, 5 HA Set 1 , or 5 HA Set 2 showed a significant difference compared to control all p values,0.001 . Among the HA-immunized groups, there was no significant difference between any two groups p.0.08 for all comparisons . For example, no significant difference was observed between the A/Anhui/1/05 group, which had the least survival among the HA immunized groups 7 out of 10 , and other HA groups: A/Indonesia/5/05 p = 0.377 , 10 HA p = 0.082 , 5 HA Set 1 p = 0.101 , or 5 HA Set 2 p = .411 . Therefore, we cannot exclude the possibility that the 3 deaths in the A/Anhui/1/05 group may have been due to random chance. Since it is desirable to confer protective immunity in poultry and HA DNA vaccination was effective in mice, we next examined the breadth and potency of single or multiple HA plasmid immunization in chickens. The ability of chickens to generate specific antibodies was assessed with three strains that showed broad cross protection in mouse studies A/Vietnam/1203/2004, A/Anhui/ 1/2005 and A/Indonesia/05/2005 , administered individually or in combination, by different injection methods. In addition to needle injection, a needle-free repetitive injection device, Agro-JetH Medical International Technology, Inc., Denver, CO , was analyzed. This device disperses the 0.1 to 5 ml injection doses into the dermal, subcutaneous, or intramuscular tissue depending upon the pressure adjustments, powered by a CO 2 gas pressure plunger . The injection conditions were determined by histologic analysis of tissues that received injections of India ink; a pressure of 48 psi was chosen since it enabled consistent delivery into intradermal and subcutaneous tissues Fig. S1 . Immunization of chickens with the control plasmid CMV/R without an HA gene insert elicited minimal neutralizing antibody titers compared to HA-immunized animals 1 week after 3 DNA immunizations. Nearly all chickens immunized with either monovalent or multivalent HA DNA vaccines generated significant neutralization titers Fig. 4 and Table S1 . In general, there was a progressive increase in the amount of neutralization after each successive DNA vaccination data not shown with maximal response at 1 week after the 3 rd DNA immunization, with highest and most consistent levels in the trivalent vaccine group delivered with the Agro-JetH device. Neutralization of the Indonesia HA strain was the most robust, with neutralization nearing 100% at titers greater than 1:3200. Both the monovalent and multivalent vaccines elicited robust homologous vaccine Fig. 4 . Even though one chicken . in the multivalent vaccine group produced almost the same degree of neutralization at each time point and was protected, it did not produce a high neutralizing antibody titer for reasons that were uncertain but possibly related to a non-specific inhibitor in the sera. To determine whether chickens immunized with single or multiple DNA vaccines were protected from a lethal challenge of a heterologous HPAI H5N1 virus, vaccinated chickens were In panels B and C, mice n = 10 were immunized with 15 mg of plasmid 3 mg each three times at 3 week intervals. Serum pools from the immunized animals were collected 14 days after the third immunization. The antisera were tested against the 12 indicated pseudotyped lentiviral vectors at varying dilutions. Error bars at each point indicate the standard deviation; each sample was evaluated in triplicate. In general, the immunized serum neutralized all tested pseudotyped lentiviruses at low dilutions while differences were often observed at high dilution. .1371/journal.pone.0002432.g002 inoculated with 20 LD 50 of highly pathogenic A/Vietnam/1203/ 2004 heterologous virus intranasally using standard methods and monitored for morbidity, mortality, viral shedding and serum antibodies. While all the control animals died within 2 days of infection, 100% survival was noted in the rest of the chickens Fig. 5A . The animals that were healthy, showing no signs of clinical disease or malaise, were euthanized on day 14. There was no evidence for viral shedding monitored via tracheal and cloacal swabs of infected chickens 2-14 days after challenge as determined by embryonal inoculation data not shown: egg infectious dose 50 EID 50 limit of detection ,100 virus particles . To compare the relative efficacy of DNA vaccines delivered IM by needle and syringe versus the needle-free Agro-JetH device injection, a dose-response study was performed with amounts of DNA vaccine ranging from 500 to 0.5 mg with two inoculations. In these experiments, the HA derived from A/chicken/Nigeria/641/ 2006 was substituted for A/Vietnam/1203/2004 since it represented a more contemporary isolate. The observed rate of protection was higher among the animals receiving 5 mg by Agro-Jet 8/8 than by IM injection 6/8 Fig. 5, B vs. C . Both modes provided complete protection for all animals at doses higher than this, and 25% protection for the animals receiving 0.5 mg doses Fig. 5B, C . Survival differences between consecutive doses were assessed using a log-rank test on the survival curves for pairs of groups. A test was deemed significant if the p-value was ,.01, and marginally significant if the p-value was ,.05 but ..01. Chickens injected IM showed a marginally significant difference between 0.5 and 5 mg p = .047 . In the same group there was a significant difference between control and 5, 50 and 500 mg p,.001 for all comparisons and the difference between control and 0.5 mg was marginally significant p = .016 . Chickens that were injected using Agro-JetH showed a significant difference between 0.5 and 5 mg p = .004 and between control and 5, 50, and 500 mg p,.001 for all comparisons . There were no differences between control and 0.5 mg or between 5, 50, and 500 mg. Lastly, the survival differences between Agro-JetH and IM for each dose group were not significant. The neutralizing antibody response to homologous and heterologous HAs corresponded with protection and correlated with dose, with higher titers elicited by injection with Agro-JetH compared to needle Table S2 . We assessed viable viral shedding after inoculation by chick embryo inoculation three days after virus challenge Week 8 . While we noted some embryonic lethality at the 0.5 mg dose, there was no embryonic lethality at 5, 50 or 500 mg groups data not shown . Since the HPAI H5N1 virus first appeared ten years ago, this highly pathogenic avian influenza virus has shown increasing diversification and dissemination in Asia, Africa, and Europe 28, . In addition to its effects on human health by crossspecies transmission and ability to compromise food sources, it poses a continuing threat to public health as it evolves and adapts in different species. The pandemic potential of this virus, especially as it relates to the poultry industry and for reservoir avian hosts, underscores the need for a vaccine that offers broad spectrum immunity and protection against lethal viral challenge. While the virus remains restricted in its ability to infect humans and undergo efficient human-to-human transmission , its persistence and spread in poultry increases the risk of the emergence of a pandemic strain. One approach to pandemic risk reduction is to limit the propagation of the virus in poultry and other relevant avian species. We have previously reported that DNA vaccines encoding HA can confer protection against a highly lethal human pandemic influenza virus, the 1918 H1N1 virus, in mice . DNA vaccines offer several advantages, including the ability to express diverse antigens, tolerability in various hosts, ease of delivery, and stability for storage and distribution without the necessity of maintaining a cold chain; they have been shown to be safe and efficacious in a variety of animal models . Because they do not contain other viral proteins used to screen for infection, they also address the need to differentiate vaccinated from infected animals. There is evidence that DNA vaccination elicits cell-mediated immunity against influenza HA in addition to inducing an antibody response , an effect that could significantly contribute to protective immunity as viruses show genetic drift and reduced susceptibility to neutralization. Ideally, a highly effective influenza vaccine should not only be able to let the host develop a protective immune response against a matching live virus challenge but also elicit robust protective immune responses against a broad range of homologous and heterologous H5 influenza strains. A multivalent H5 vaccine containing diverse serotypes could expand the antigenic breadth sufficiently to provide protection against heterologous challenge and may preclude the emergence of vaccine-resistant strains that may arise due to evolutionary vaccine pressure on the virus. Due to the antigenic drift and shift of the influenza virus genome, it has been very difficult to predict the next dominant strain of an avian endemic outbreak. DNA vaccines can be synthesized in a relatively short period of time, and the targeted mutations can be tailored to specific viral serotypes. The mutations promote a focused and enhanced immune response that may be particularly important in the event of an outbreak where specificity is the key to epidemic control. The use of modified codons ensures maximal expression in the host and eliminates the possibility of recombination with influenza viruses that might potentially generate new strains. A more broadly protective murine vaccine was developed here by including more HAs from varying strains in the multivalent vaccine Figs. 2 and 3 . However, it is less practical to include large numbers of different HAs in one vaccine due to the cost and complexity of manufacturing such a vaccine. Therefore, we simplified the vaccine regimen based on cross-neutralization studies and phylogenetic relationships. A trivalent vaccine was subsequently identified for further studies. Due . While three DNA immunizations were used initially to demonstrate protective immunity and have been used previously to elicit protection in mice , we found that effective protective immunity could be induced with two DNA vaccinations and as little as 5 mg trivalent DNA immunization using the ID/SC route with the Agro-JetH device. In addition, based on the chick embryo inoculation data, we believe that there is effective neutralization of the virus and lack of infectious viral shedding in chicken vaccinated with as little as 5 mg of DNA. The device's capacity for rapid repetitive injection and the lower quantity and stability of DNA enhance the practicality and utility of this approach for vaccination of endangered species in captivity or administration to poultry or other animals. A/Vietnam/1203/2004 H5N1 A/VN/1203/04 was obtained from the repository at the Centers for Disease Control and Prevention CDC , Atlanta, Georgia. The virus was propagated in 10-day old embryonated chicken eggs at 35uC and stored at 270uC until use. The virus was titrated by the Reed and Muench method to determine EID 50 . GenBank ABD28180 were synthesized using human-preferred codons GeneArt, Regensburg, Germany . HA cDNAs from diverse strains of influenza viruses were then inserted into plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo . For initial trivalent immunizations in chickens, the A/Vietnam/1203/ 2004, A/Anhui/1/2005 and A/Indonesia/05/2005 strains were used and in the dose response study, the Vietnam strain was replaced with A/chicken/Nigeria/641/2006. The immunogens used in DNA vaccination contained a cleavage site mutation PQRERRRKKRG to PQRETRG as previously described . This mutation was generated by site-directed mutagenesis using a QuickChange kit Stratagene, La Jolla, CA . DNA immunization of mice week old female BALB/c mice were purchased from The Jackson Laboratory and maintained in the AAALAC-accredited Vaccine Research Center Animal Care Facility Bethesda, MD under specific pathogen-free conditions. All experiments were approved by the Vaccine Research Center Animal Care and Use Committee. The mice were immunized as previously described . Briefly, mice 10 animals for all test groups, 20 animals for the The study was carried out in the AAALAC-accredited animal facility at the University of Maryland School of Medicine. Six groups of 8 one-day-old male and female SPAFAS White Leghorn Chickens, Gallus domesticus, were obtained from Charles River Laboratories Connecticut . The animals were housed in brooder and grower cages McMurray Hatcheries, Iowa . Feed Teklad Japanese Quail Diet -3050, Harlan-Teklad, WI and water were provided to the animals ad libitum. The study was performed in strict accordance with the ''Guide'' after approvals from the Animal Care and Use Committees of the Vaccine Research Center, NIH and the University of Maryland. DNA immunizations were performed as described at 0, 3 and 6 weeks. A total dose of 500 mg of one or a combination of the following DNA plasmids in a volume of 250 ml was administered to each animal: pCMV/ R, pCMV/R-HA Agro-JetH is a needle-free device used for mass delivery of vaccines and drugs in livestock and poultry. The device is semiautomatic and requires a small CO 2 tank or compressed air for low pressure delivery. Upon trigger activation, CO 2 disperses the injectate at a precise dose into the muscle, dermis or subcutaneous tissue depending on the setting that was standardized for our use. We used an effective volume of 0.1 ml in our injectate . In this study we were able to effectively deliver 0.1 ml of injectate into the animal's dermis/subcutaneous tissue at a pressure of 48-55 psi. Sixty-eight weeks after the last immunization, female BALB/c mice were lightly anesthetized with Ketamine/Xylazine and inoculated intranasally with 10 LD 50 of A/Vietnam/1203/2004 virus diluted in phosphate-buffered saline in a 50 ul volume. Mice were monitored daily for morbidity and measured for weight loss and mortality for 21 days post infection. Any mouse that had lost more than 25% of its body weight was euthanized. All experiments involving the HPAI virus were conducted in an AAALAC accredited facility BioQual Inc., Gaithersburg, MD under BSL 3 conditions that included enhancements required by the USDA and the Select Agent Program. White Leghorn chickens were challenged one week after the last immunization with 20 lethal dose 50 LD 50 of A/Vietnam/1203/04 H5N1 influenza A virus, equivalent to 2610 4 EID 50 based on previous challenges . Chickens were infected with 200 ml virus intranasally. Tracheal and cloacal swabs were collected days 3 and 5 post-challenge and stored in glass vials containing BHI medium BBL TM Brain Heart Infusion, Becton Dickinson at 280uC. Blood was collected 14 days post-challenge and serum was titered by microneutralization assay. Chickens were observed and scored daily for clinical signs of infection, morbidity and mortality. Chickens that survived the study were bled and humanely euthanized at day 14 post-challenge. Lungs, heart, intestine and kidney were collected and samples were stored in formalin for histopathology. Experiments were carried out under BSL3+ conditions with investigators wearing appropriate protective equipment and compliant with all Institutional Animal Care and Use Committee-approved protocols and under Animal Welfare Act regulations at the University of Maryland, College Park, Maryland. Representative tracheal and cloacal swabs were chosen to run an EID 50 assay for comparison and virus titers were determine by the method of Reed and Meunch . Briefly, swabs were used to infect 10 day-old embryonated chicken eggs in 10-fold dilutions. Three eggs were inoculated per dilution and incubated for 48 hours before titration. Neutralizing antibodies were titrated from serum samples collected week 5 and 7 post-vaccination and day 14 post-challenge. The microneutralization assay was performed using a 96-well plate format. Serum was treated with receptor-destroying enzyme Denka Seiken Co. and treated at 37uC per the manufacturer's instructions. After an overnight incubation and subsequent inactivation samples were brought to a final dilution of 1:10 using PBS and each sample was serially diluted and virus, diluted to 100 TCID 50 , was added to each well. The plates were then incubated at 37uC, 5% CO 2 for 1-2 hours. Following incubation, supernatants were used to infect a second 96-well plate of MDCK cells. Microplates were incubated at 4uC for 15 minutes and then 37uC, 5% CO 2 for 45 minutes. Supernatants of serum and virus were then discarded and 200 ml of OptiMEM containing 1X antibiotics/antimycotics, 1 mg/ml TPCK-trypsin was added and incubated at 37uC, 5% CO 2 for 3 days. After 3 days, 50 ml of the supernatant from each well was transferred into a new 96-well microplate, and an HA assay was performed to calculate the antibody titers. Virus and cell controls were included in the assay. Two-fold dilutions of heat-inactivated sera were tested in a microneutralization assay as previously described for the presence of antibodies that neutralized the infectivity of 100 TCID 50 50% tissue culture infectious dose of the A/Vietnam/ 1203/2004 H5N1 virus on MDCK cell monolayers by using two wells per dilution on a 96-well plate. The recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described . For the neutralization assay, antisera from immunized animals were heat-inactivated at 55uC for 30 minutes and mixed with 50 ml of pseudovirus at various dilutions. The sera/virus mixture was then added to 293A cells in 96-well B&W TC Isoplates Wallac, Turku, Finland; 12,000 cells/well . Two hours later, the plates were washed and fresh medium was added. Cells were lysed in mammalian cell lysis buffer Promega, Madison, WI 24 hrs after infection and luciferase activity was measured using the Luciferase Assay System Promega, Madison, WI . The following strains were used for the production of pseudotyped viruses: for HA we used A/Thailand/1 KAN- The HA/HI titers were determined as previously described . Briefly, HA titers were calculated using 50 ml of 0.5% chicken red blood cell suspension in PBS added to 50 ml of twofold dilutions of virus in PBS. This mix was incubated at room temperature for 30 minutes. The HA titers were calculated as the reciprocal value of the highest dilution that caused complete hemagglutination. HI titers were calculated by titrating 50 ml of antiserum treated with receptor-destroying enzyme and an equivalent amount of A/Vietnam/1203/2004 virus four hemagglutinating doses was added to each well. Wells were incubated at room temperature for 30 minutes and 50 ml of a 0.5% suspension of chicken red blood cells was added. HI titers were calculated after 30 minutes as the reciprocal of the serum dilution that inhibited hemagglutination. Table S1 Hemagglutination inhibition HI , microneutralization titer NT , and LAI of sera from individual chickens immunized with different vaccines. Sera from immunized animals were obtained at week 5 or 7, a week before or after the final boost, and neutralization was assessed by HI, microneutralization NT and LAI shown as IC 50 . Individual animal serum of each group is shown and was analyzed as described in the Materials and Methods section. Figure S1 Characterization of needle-free Agro-JetH DNA immunization in chickens. To evaluate the distribution of fluid into superficial or deep layers of subcutaneous tissues after delivery by AgroJetH, 4 or 7 week old chickens were injected with a solution containing India ink with this needle-free device at various pressures, ranging from 45 to 55 mm Hg. Three sites thigh, wing and breast were used, and biopsies were taken for routine hematoxylin and eosin staining. Representative sections of thigh injections are shown from 7 week old chickens and were similar at 4 weeks data not shown . While the 48 mm Hg pressure deposited the injectate into the dermis/subcutaneous region left , the higher pressure injections, 52 and 58 mm Hg, deposited the injectate into the subcutaneous and muscle layers middle, right . 48 mm Hg consistently provided an optimal pressure to deposit the injectate into the dermis and subcutaneous tissue and was chosen for all AgroJetH immunizations. Found at: .1371/journal.pone.0002432.s003 10.74 MB DOC
What is the relationship between urbanization and risk of emergence of flu-like diseases?	findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk.	[ "Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases EID , the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza HPAI H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas.", "Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances .", "These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances . Highly Pathogenic Avian Influenza HPAI subtype H5N1 is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of \"surprise\" . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015.", "In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility . Lederberg et al. first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers.", "first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial \"adaptation and change,\" along with \"changing ecosystems\" and \"economic development and land use\" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying \"Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants humans included of these environments, and our interactions including hygienic and therapeutic interventions with the parasites\" . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the \"emerging properties\" of complex adaptive systems CAS .", "Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the \"emerging properties\" of complex adaptive systems CAS . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely.", "The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1 peri-urban areas, as defined by Saksena et al. , 2 land-use diversity, and 3 co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years .", "We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time . but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities.", "but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes rural areas and towns, and provincial towns and cities are divided into wards urban subdistricts and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term \"commune\" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work.", "For the purpose of simplicity we will henceforth use the term \"commune\" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health.", "Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban .", "Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban . Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness . Other studies prioritized human population density as a satisfactory surrogate 11, , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI.", "Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of \"ruralness\". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for \"village-town\" .", "While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of \"ruralness\". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for \"village-town\" . McGee identified six characteristics of desakota regions: 1 a large population of smallholder cultivators; 2 an increase in non-agricultural activities; 3 extreme fluidity and mobility of population; 4 a mixture of land uses, agriculture, cottage industries, suburban development; 5 increased participation of the female labor force; and 6 \"grey-zones\", where informal and illegal activities group . Saksena et al. built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core.", "built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes LCLUC . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease .", "LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts , although it is not clear if reduced species diversity necessarily increases pathogen transmission . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere.", "Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 , much of this demand is met by farms near cities , many in areas undergoing processes of peri-urbanization . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers . Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds.", "Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive backyard systems and intensive systems 15, 37 . In much of Asia there is often a mix of commercial and backyard farming at any one location . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor . Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation .", "Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. 15, 37 . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes.", "In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project.", "Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI .", "to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI . The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero.", "The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land including miscellaneous uses for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles.", "The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density 11, 14-16, 18, 19, 44, 45 . • Poultry trade and market. Towns and cities were assumed to be active trading places . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure .", "So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure . So, the distance to the nearest a national highway and b provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area . • Intermediate hosts. Duck and geese densities were calculated using total commune area . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors.", "As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus.", "We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks 47, , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk .", "We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam . Compound Topographical Index CTI, also known as Topographical Wetness Index is a measure of the tendency for water to pool. Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy.", "Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models ENM of HPAI H5N1 . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance .", "CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln A s / tan β where; A s = Area Value calculated as flow accumulation + 1 Ã pixel area in m 2 and β is the slope expressed in radians . Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health .", "Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune.", "In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose.", "Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually.", "We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level.", "Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI .", "This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI . Compound Topographical Index CTI data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation .", "Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation . Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors or their log-transform, as the case may be . Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient .", "Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient . When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term .", "Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time.", "The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 in at least one wave-place combination and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies . We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects.", "We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology . It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, .", "It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model.", "We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate lr and tree complexity tc to determine the number of trees for optimal prediction . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed.", "Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve AUC of the receiver operation characteristic ROC curve. AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates .", "AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates . We used the corrected Akaike Information Criteria AICc to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 IBM Corp., New York, 2012 for GLMM and R version 3.1.0 The R Foundation for Statistical Computing, 2014 for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations.", "For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 Tables 2-7 . The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level.", "The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 AUC = 0.69 and Wave 2 AUC = 0.77 by Gilbert et al. . Both Gilbert et al. and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta.", "and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions .", "The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk RR of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f .", "In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2.", "Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1.", "This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places.", "Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher 60-78% than at the delta levels 14-35% . In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance.", "In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable poultry outbreaks is that the data may have reporting/detection biases . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose.", "We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry.", "This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1 when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2 high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3 landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al.", "Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1.", "When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity.", "Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics e.g. . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g.", "This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g. 15, . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity .", "Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity . Mapping land-use classes at the national scale for local administrative units i.e., the 10,820 communes in Viet Nam is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited.", "Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level.", "So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors.", "CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant.", "Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere.", "The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar.", "Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes.", "Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices." ]	1,628	585	Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases EID , the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza HPAI H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances . Highly Pathogenic Avian Influenza HPAI subtype H5N1 is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility . Lederberg et al. first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants humans included of these environments, and our interactions including hygienic and therapeutic interventions with the parasites" . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems CAS . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1 peri-urban areas, as defined by Saksena et al. , 2 land-use diversity, and 3 co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time . but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes rural areas and towns, and provincial towns and cities are divided into wards urban subdistricts and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban . Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness . Other studies prioritized human population density as a satisfactory surrogate 11, , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for "village-town" . McGee identified six characteristics of desakota regions: 1 a large population of smallholder cultivators; 2 an increase in non-agricultural activities; 3 extreme fluidity and mobility of population; 4 a mixture of land uses, agriculture, cottage industries, suburban development; 5 increased participation of the female labor force; and 6 "grey-zones", where informal and illegal activities group . Saksena et al. built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes LCLUC . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts , although it is not clear if reduced species diversity necessarily increases pathogen transmission . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 , much of this demand is met by farms near cities , many in areas undergoing processes of peri-urbanization . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers . Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive backyard systems and intensive systems 15, 37 . In much of Asia there is often a mix of commercial and backyard farming at any one location . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor . Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. 15, 37 . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI . The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land including miscellaneous uses for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density 11, 14-16, 18, 19, 44, 45 . • Poultry trade and market. Towns and cities were assumed to be active trading places . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure . So, the distance to the nearest a national highway and b provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area . • Intermediate hosts. Duck and geese densities were calculated using total commune area . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks 47, , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam . Compound Topographical Index CTI, also known as Topographical Wetness Index is a measure of the tendency for water to pool. Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models ENM of HPAI H5N1 . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln A s / tan β where; A s = Area Value calculated as flow accumulation + 1 Ã pixel area in m 2 and β is the slope expressed in radians . Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI . Compound Topographical Index CTI data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation . Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors or their log-transform, as the case may be . Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient . When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 in at least one wave-place combination and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies . We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology . It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate lr and tree complexity tc to determine the number of trees for optimal prediction . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve AUC of the receiver operation characteristic ROC curve. AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates . We used the corrected Akaike Information Criteria AICc to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 IBM Corp., New York, 2012 for GLMM and R version 3.1.0 The R Foundation for Statistical Computing, 2014 for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 Tables 2-7 . The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 AUC = 0.69 and Wave 2 AUC = 0.77 by Gilbert et al. . Both Gilbert et al. and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk RR of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher 60-78% than at the delta levels 14-35% . In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable poultry outbreaks is that the data may have reporting/detection biases . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1 when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2 high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3 landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics e.g. . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g. 15, . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity . Mapping land-use classes at the national scale for local administrative units i.e., the 10,820 communes in Viet Nam is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.
What factors and characteristics of semi-urban landscapes promote viral transmission?	higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission	[ "Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases EID , the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza HPAI H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas.", "Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances .", "These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances . Highly Pathogenic Avian Influenza HPAI subtype H5N1 is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of \"surprise\" . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015.", "In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility . Lederberg et al. first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers.", "first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial \"adaptation and change,\" along with \"changing ecosystems\" and \"economic development and land use\" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying \"Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants humans included of these environments, and our interactions including hygienic and therapeutic interventions with the parasites\" . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the \"emerging properties\" of complex adaptive systems CAS .", "Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the \"emerging properties\" of complex adaptive systems CAS . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely.", "The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1 peri-urban areas, as defined by Saksena et al. , 2 land-use diversity, and 3 co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years .", "We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time . but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities.", "but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes rural areas and towns, and provincial towns and cities are divided into wards urban subdistricts and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term \"commune\" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work.", "For the purpose of simplicity we will henceforth use the term \"commune\" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health.", "Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban .", "Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban . Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness . Other studies prioritized human population density as a satisfactory surrogate 11, , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI.", "Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of \"ruralness\". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for \"village-town\" .", "While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of \"ruralness\". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for \"village-town\" . McGee identified six characteristics of desakota regions: 1 a large population of smallholder cultivators; 2 an increase in non-agricultural activities; 3 extreme fluidity and mobility of population; 4 a mixture of land uses, agriculture, cottage industries, suburban development; 5 increased participation of the female labor force; and 6 \"grey-zones\", where informal and illegal activities group . Saksena et al. built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core.", "built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes LCLUC . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease .", "LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts , although it is not clear if reduced species diversity necessarily increases pathogen transmission . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere.", "Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 , much of this demand is met by farms near cities , many in areas undergoing processes of peri-urbanization . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers . Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds.", "Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive backyard systems and intensive systems 15, 37 . In much of Asia there is often a mix of commercial and backyard farming at any one location . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor . Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation .", "Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. 15, 37 . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes.", "In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project.", "Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI .", "to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI . The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero.", "The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land including miscellaneous uses for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles.", "The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density 11, 14-16, 18, 19, 44, 45 . • Poultry trade and market. Towns and cities were assumed to be active trading places . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure .", "So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure . So, the distance to the nearest a national highway and b provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area . • Intermediate hosts. Duck and geese densities were calculated using total commune area . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors.", "As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus.", "We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks 47, , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk .", "We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam . Compound Topographical Index CTI, also known as Topographical Wetness Index is a measure of the tendency for water to pool. Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy.", "Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models ENM of HPAI H5N1 . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance .", "CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln A s / tan β where; A s = Area Value calculated as flow accumulation + 1 Ã pixel area in m 2 and β is the slope expressed in radians . Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health .", "Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune.", "In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose.", "Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually.", "We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level.", "Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI .", "This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI . Compound Topographical Index CTI data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation .", "Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation . Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors or their log-transform, as the case may be . Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient .", "Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient . When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term .", "Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time.", "The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 in at least one wave-place combination and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies . We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects.", "We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology . It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, .", "It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model.", "We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate lr and tree complexity tc to determine the number of trees for optimal prediction . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed.", "Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve AUC of the receiver operation characteristic ROC curve. AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates .", "AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates . We used the corrected Akaike Information Criteria AICc to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 IBM Corp., New York, 2012 for GLMM and R version 3.1.0 The R Foundation for Statistical Computing, 2014 for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations.", "For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 Tables 2-7 . The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level.", "The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 AUC = 0.69 and Wave 2 AUC = 0.77 by Gilbert et al. . Both Gilbert et al. and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta.", "and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions .", "The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk RR of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f .", "In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2.", "Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1.", "This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places.", "Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher 60-78% than at the delta levels 14-35% . In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance.", "In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable poultry outbreaks is that the data may have reporting/detection biases . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose.", "We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry.", "This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1 when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2 high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3 landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al.", "Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1.", "When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity.", "Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics e.g. . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g.", "This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g. 15, . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity .", "Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity . Mapping land-use classes at the national scale for local administrative units i.e., the 10,820 communes in Viet Nam is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited.", "Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level.", "So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors.", "CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant.", "Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere.", "The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar.", "Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes.", "Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices." ]	1,628	586	Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases EID , the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza HPAI H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances . Highly Pathogenic Avian Influenza HPAI subtype H5N1 is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility . Lederberg et al. first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants humans included of these environments, and our interactions including hygienic and therapeutic interventions with the parasites" . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems CAS . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1 peri-urban areas, as defined by Saksena et al. , 2 land-use diversity, and 3 co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time . but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes rural areas and towns, and provincial towns and cities are divided into wards urban subdistricts and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban . Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness . Other studies prioritized human population density as a satisfactory surrogate 11, , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for "village-town" . McGee identified six characteristics of desakota regions: 1 a large population of smallholder cultivators; 2 an increase in non-agricultural activities; 3 extreme fluidity and mobility of population; 4 a mixture of land uses, agriculture, cottage industries, suburban development; 5 increased participation of the female labor force; and 6 "grey-zones", where informal and illegal activities group . Saksena et al. built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes LCLUC . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts , although it is not clear if reduced species diversity necessarily increases pathogen transmission . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 , much of this demand is met by farms near cities , many in areas undergoing processes of peri-urbanization . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers . Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive backyard systems and intensive systems 15, 37 . In much of Asia there is often a mix of commercial and backyard farming at any one location . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor . Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. 15, 37 . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI . The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land including miscellaneous uses for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density 11, 14-16, 18, 19, 44, 45 . • Poultry trade and market. Towns and cities were assumed to be active trading places . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure . So, the distance to the nearest a national highway and b provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area . • Intermediate hosts. Duck and geese densities were calculated using total commune area . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks 47, , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam . Compound Topographical Index CTI, also known as Topographical Wetness Index is a measure of the tendency for water to pool. Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models ENM of HPAI H5N1 . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln A s / tan β where; A s = Area Value calculated as flow accumulation + 1 Ã pixel area in m 2 and β is the slope expressed in radians . Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI . Compound Topographical Index CTI data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation . Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors or their log-transform, as the case may be . Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient . When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 in at least one wave-place combination and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies . We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology . It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate lr and tree complexity tc to determine the number of trees for optimal prediction . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve AUC of the receiver operation characteristic ROC curve. AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates . We used the corrected Akaike Information Criteria AICc to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 IBM Corp., New York, 2012 for GLMM and R version 3.1.0 The R Foundation for Statistical Computing, 2014 for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 Tables 2-7 . The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 AUC = 0.69 and Wave 2 AUC = 0.77 by Gilbert et al. . Both Gilbert et al. and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk RR of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher 60-78% than at the delta levels 14-35% . In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable poultry outbreaks is that the data may have reporting/detection biases . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1 when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2 high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3 landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics e.g. . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g. 15, . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity . Mapping land-use classes at the national scale for local administrative units i.e., the 10,820 communes in Viet Nam is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.
What is the relationship between HIN1 viral transmission and poultry production.	landscapes where intensive and extensive forms of poultry production overlap were found at greater risk	[ "Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases EID , the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza HPAI H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas.", "Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances .", "These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances . Highly Pathogenic Avian Influenza HPAI subtype H5N1 is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of \"surprise\" . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015.", "In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility . Lederberg et al. first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers.", "first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial \"adaptation and change,\" along with \"changing ecosystems\" and \"economic development and land use\" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying \"Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants humans included of these environments, and our interactions including hygienic and therapeutic interventions with the parasites\" . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the \"emerging properties\" of complex adaptive systems CAS .", "Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the \"emerging properties\" of complex adaptive systems CAS . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely.", "The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1 peri-urban areas, as defined by Saksena et al. , 2 land-use diversity, and 3 co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years .", "We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time . but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities.", "but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes rural areas and towns, and provincial towns and cities are divided into wards urban subdistricts and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term \"commune\" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work.", "For the purpose of simplicity we will henceforth use the term \"commune\" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health.", "Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban .", "Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban . Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness . Other studies prioritized human population density as a satisfactory surrogate 11, , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI.", "Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of \"ruralness\". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for \"village-town\" .", "While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of \"ruralness\". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for \"village-town\" . McGee identified six characteristics of desakota regions: 1 a large population of smallholder cultivators; 2 an increase in non-agricultural activities; 3 extreme fluidity and mobility of population; 4 a mixture of land uses, agriculture, cottage industries, suburban development; 5 increased participation of the female labor force; and 6 \"grey-zones\", where informal and illegal activities group . Saksena et al. built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core.", "built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes LCLUC . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease .", "LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts , although it is not clear if reduced species diversity necessarily increases pathogen transmission . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere.", "Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 , much of this demand is met by farms near cities , many in areas undergoing processes of peri-urbanization . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers . Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds.", "Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive backyard systems and intensive systems 15, 37 . In much of Asia there is often a mix of commercial and backyard farming at any one location . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor . Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation .", "Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. 15, 37 . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes.", "In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project.", "Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI .", "to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI . The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero.", "The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land including miscellaneous uses for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles.", "The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density 11, 14-16, 18, 19, 44, 45 . • Poultry trade and market. Towns and cities were assumed to be active trading places . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure .", "So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure . So, the distance to the nearest a national highway and b provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area . • Intermediate hosts. Duck and geese densities were calculated using total commune area . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors.", "As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus.", "We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks 47, , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk .", "We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam . Compound Topographical Index CTI, also known as Topographical Wetness Index is a measure of the tendency for water to pool. Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy.", "Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models ENM of HPAI H5N1 . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance .", "CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln A s / tan β where; A s = Area Value calculated as flow accumulation + 1 Ã pixel area in m 2 and β is the slope expressed in radians . Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health .", "Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune.", "In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose.", "Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually.", "We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level.", "Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI .", "This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI . Compound Topographical Index CTI data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation .", "Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation . Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors or their log-transform, as the case may be . Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient .", "Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient . When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term .", "Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time.", "The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 in at least one wave-place combination and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies . We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects.", "We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology . It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, .", "It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model.", "We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate lr and tree complexity tc to determine the number of trees for optimal prediction . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed.", "Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve AUC of the receiver operation characteristic ROC curve. AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates .", "AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates . We used the corrected Akaike Information Criteria AICc to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 IBM Corp., New York, 2012 for GLMM and R version 3.1.0 The R Foundation for Statistical Computing, 2014 for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations.", "For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 Tables 2-7 . The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level.", "The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 AUC = 0.69 and Wave 2 AUC = 0.77 by Gilbert et al. . Both Gilbert et al. and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta.", "and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions .", "The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk RR of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f .", "In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2.", "Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1.", "This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places.", "Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher 60-78% than at the delta levels 14-35% . In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance.", "In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable poultry outbreaks is that the data may have reporting/detection biases . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose.", "We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry.", "This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1 when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2 high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3 landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al.", "Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1.", "When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity.", "Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics e.g. . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g.", "This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g. 15, . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity .", "Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity . Mapping land-use classes at the national scale for local administrative units i.e., the 10,820 communes in Viet Nam is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited.", "Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level.", "So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors.", "CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant.", "Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere.", "The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar.", "Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes.", "Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices." ]	1,628	587	Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases EID , the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza HPAI H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances . Highly Pathogenic Avian Influenza HPAI subtype H5N1 is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility . Lederberg et al. first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants humans included of these environments, and our interactions including hygienic and therapeutic interventions with the parasites" . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems CAS . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1 peri-urban areas, as defined by Saksena et al. , 2 land-use diversity, and 3 co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time . but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes rural areas and towns, and provincial towns and cities are divided into wards urban subdistricts and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban . Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness . Other studies prioritized human population density as a satisfactory surrogate 11, , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for "village-town" . McGee identified six characteristics of desakota regions: 1 a large population of smallholder cultivators; 2 an increase in non-agricultural activities; 3 extreme fluidity and mobility of population; 4 a mixture of land uses, agriculture, cottage industries, suburban development; 5 increased participation of the female labor force; and 6 "grey-zones", where informal and illegal activities group . Saksena et al. built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes LCLUC . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts , although it is not clear if reduced species diversity necessarily increases pathogen transmission . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 , much of this demand is met by farms near cities , many in areas undergoing processes of peri-urbanization . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers . Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive backyard systems and intensive systems 15, 37 . In much of Asia there is often a mix of commercial and backyard farming at any one location . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor . Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. 15, 37 . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI . The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land including miscellaneous uses for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density 11, 14-16, 18, 19, 44, 45 . • Poultry trade and market. Towns and cities were assumed to be active trading places . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure . So, the distance to the nearest a national highway and b provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area . • Intermediate hosts. Duck and geese densities were calculated using total commune area . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks 47, , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam . Compound Topographical Index CTI, also known as Topographical Wetness Index is a measure of the tendency for water to pool. Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models ENM of HPAI H5N1 . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln A s / tan β where; A s = Area Value calculated as flow accumulation + 1 Ã pixel area in m 2 and β is the slope expressed in radians . Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI . Compound Topographical Index CTI data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation . Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors or their log-transform, as the case may be . Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient . When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 in at least one wave-place combination and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies . We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology . It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate lr and tree complexity tc to determine the number of trees for optimal prediction . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve AUC of the receiver operation characteristic ROC curve. AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates . We used the corrected Akaike Information Criteria AICc to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 IBM Corp., New York, 2012 for GLMM and R version 3.1.0 The R Foundation for Statistical Computing, 2014 for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 Tables 2-7 . The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 AUC = 0.69 and Wave 2 AUC = 0.77 by Gilbert et al. . Both Gilbert et al. and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk RR of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher 60-78% than at the delta levels 14-35% . In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable poultry outbreaks is that the data may have reporting/detection biases . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1 when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2 high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3 landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics e.g. . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g. 15, . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity . Mapping land-use classes at the national scale for local administrative units i.e., the 10,820 communes in Viet Nam is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.
What is the principle behind infection Convergence Model ?	The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence.	[ "Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases EID , the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza HPAI H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas.", "Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances .", "These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances . Highly Pathogenic Avian Influenza HPAI subtype H5N1 is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of \"surprise\" . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015.", "In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility . Lederberg et al. first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers.", "first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial \"adaptation and change,\" along with \"changing ecosystems\" and \"economic development and land use\" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying \"Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants humans included of these environments, and our interactions including hygienic and therapeutic interventions with the parasites\" . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the \"emerging properties\" of complex adaptive systems CAS .", "Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the \"emerging properties\" of complex adaptive systems CAS . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely.", "The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1 peri-urban areas, as defined by Saksena et al. , 2 land-use diversity, and 3 co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years .", "We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time . but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities.", "but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes rural areas and towns, and provincial towns and cities are divided into wards urban subdistricts and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term \"commune\" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work.", "For the purpose of simplicity we will henceforth use the term \"commune\" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health.", "Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban .", "Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban . Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness . Other studies prioritized human population density as a satisfactory surrogate 11, , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI.", "Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of \"ruralness\". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for \"village-town\" .", "While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of \"ruralness\". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for \"village-town\" . McGee identified six characteristics of desakota regions: 1 a large population of smallholder cultivators; 2 an increase in non-agricultural activities; 3 extreme fluidity and mobility of population; 4 a mixture of land uses, agriculture, cottage industries, suburban development; 5 increased participation of the female labor force; and 6 \"grey-zones\", where informal and illegal activities group . Saksena et al. built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core.", "built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes LCLUC . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease .", "LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts , although it is not clear if reduced species diversity necessarily increases pathogen transmission . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere.", "Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 , much of this demand is met by farms near cities , many in areas undergoing processes of peri-urbanization . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers . Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds.", "Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive backyard systems and intensive systems 15, 37 . In much of Asia there is often a mix of commercial and backyard farming at any one location . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor . Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation .", "Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. 15, 37 . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes.", "In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project.", "Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI .", "to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI . The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero.", "The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land including miscellaneous uses for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles.", "The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density 11, 14-16, 18, 19, 44, 45 . • Poultry trade and market. Towns and cities were assumed to be active trading places . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure .", "So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure . So, the distance to the nearest a national highway and b provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area . • Intermediate hosts. Duck and geese densities were calculated using total commune area . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors.", "As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus.", "We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks 47, , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk .", "We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam . Compound Topographical Index CTI, also known as Topographical Wetness Index is a measure of the tendency for water to pool. Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy.", "Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models ENM of HPAI H5N1 . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance .", "CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln A s / tan β where; A s = Area Value calculated as flow accumulation + 1 Ã pixel area in m 2 and β is the slope expressed in radians . Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health .", "Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune.", "In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose.", "Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually.", "We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level.", "Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI .", "This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI . Compound Topographical Index CTI data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation .", "Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation . Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors or their log-transform, as the case may be . Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient .", "Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient . When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term .", "Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time.", "The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 in at least one wave-place combination and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies . We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects.", "We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology . It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, .", "It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model.", "We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate lr and tree complexity tc to determine the number of trees for optimal prediction . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed.", "Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve AUC of the receiver operation characteristic ROC curve. AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates .", "AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates . We used the corrected Akaike Information Criteria AICc to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 IBM Corp., New York, 2012 for GLMM and R version 3.1.0 The R Foundation for Statistical Computing, 2014 for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations.", "For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 Tables 2-7 . The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level.", "The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 AUC = 0.69 and Wave 2 AUC = 0.77 by Gilbert et al. . Both Gilbert et al. and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta.", "and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions .", "The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk RR of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f .", "In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2.", "Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1.", "This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places.", "Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher 60-78% than at the delta levels 14-35% . In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance.", "In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable poultry outbreaks is that the data may have reporting/detection biases . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose.", "We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry.", "This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1 when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2 high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3 landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al.", "Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1.", "When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity.", "Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics e.g. . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g.", "This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g. 15, . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity .", "Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity . Mapping land-use classes at the national scale for local administrative units i.e., the 10,820 communes in Viet Nam is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited.", "Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level.", "So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors.", "CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant.", "Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere.", "The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar.", "Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes.", "Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices." ]	1,628	588	Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases EID , the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza HPAI H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances . Highly Pathogenic Avian Influenza HPAI subtype H5N1 is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility . Lederberg et al. first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants humans included of these environments, and our interactions including hygienic and therapeutic interventions with the parasites" . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems CAS . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1 peri-urban areas, as defined by Saksena et al. , 2 land-use diversity, and 3 co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time . but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes rural areas and towns, and provincial towns and cities are divided into wards urban subdistricts and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban . Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness . Other studies prioritized human population density as a satisfactory surrogate 11, , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for "village-town" . McGee identified six characteristics of desakota regions: 1 a large population of smallholder cultivators; 2 an increase in non-agricultural activities; 3 extreme fluidity and mobility of population; 4 a mixture of land uses, agriculture, cottage industries, suburban development; 5 increased participation of the female labor force; and 6 "grey-zones", where informal and illegal activities group . Saksena et al. built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes LCLUC . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts , although it is not clear if reduced species diversity necessarily increases pathogen transmission . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 , much of this demand is met by farms near cities , many in areas undergoing processes of peri-urbanization . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers . Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive backyard systems and intensive systems 15, 37 . In much of Asia there is often a mix of commercial and backyard farming at any one location . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor . Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. 15, 37 . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI . The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land including miscellaneous uses for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density 11, 14-16, 18, 19, 44, 45 . • Poultry trade and market. Towns and cities were assumed to be active trading places . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure . So, the distance to the nearest a national highway and b provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area . • Intermediate hosts. Duck and geese densities were calculated using total commune area . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks 47, , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam . Compound Topographical Index CTI, also known as Topographical Wetness Index is a measure of the tendency for water to pool. Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models ENM of HPAI H5N1 . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln A s / tan β where; A s = Area Value calculated as flow accumulation + 1 Ã pixel area in m 2 and β is the slope expressed in radians . Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI . Compound Topographical Index CTI data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation . Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors or their log-transform, as the case may be . Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient . When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 in at least one wave-place combination and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies . We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology . It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate lr and tree complexity tc to determine the number of trees for optimal prediction . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve AUC of the receiver operation characteristic ROC curve. AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates . We used the corrected Akaike Information Criteria AICc to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 IBM Corp., New York, 2012 for GLMM and R version 3.1.0 The R Foundation for Statistical Computing, 2014 for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 Tables 2-7 . The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 AUC = 0.69 and Wave 2 AUC = 0.77 by Gilbert et al. . Both Gilbert et al. and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk RR of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher 60-78% than at the delta levels 14-35% . In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable poultry outbreaks is that the data may have reporting/detection biases . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1 when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2 high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3 landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics e.g. . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g. 15, . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity . Mapping land-use classes at the national scale for local administrative units i.e., the 10,820 communes in Viet Nam is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.
What is the Boosted Regression Tree method?	BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model	[ "Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases EID , the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza HPAI H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas.", "Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances .", "These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances . Highly Pathogenic Avian Influenza HPAI subtype H5N1 is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of \"surprise\" . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015.", "In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility . Lederberg et al. first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers.", "first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial \"adaptation and change,\" along with \"changing ecosystems\" and \"economic development and land use\" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying \"Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants humans included of these environments, and our interactions including hygienic and therapeutic interventions with the parasites\" . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the \"emerging properties\" of complex adaptive systems CAS .", "Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the \"emerging properties\" of complex adaptive systems CAS . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely.", "The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1 peri-urban areas, as defined by Saksena et al. , 2 land-use diversity, and 3 co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years .", "We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time . but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities.", "but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes rural areas and towns, and provincial towns and cities are divided into wards urban subdistricts and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term \"commune\" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work.", "For the purpose of simplicity we will henceforth use the term \"commune\" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health.", "Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban .", "Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban . Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness . Other studies prioritized human population density as a satisfactory surrogate 11, , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI.", "Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of \"ruralness\". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for \"village-town\" .", "While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of \"ruralness\". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for \"village-town\" . McGee identified six characteristics of desakota regions: 1 a large population of smallholder cultivators; 2 an increase in non-agricultural activities; 3 extreme fluidity and mobility of population; 4 a mixture of land uses, agriculture, cottage industries, suburban development; 5 increased participation of the female labor force; and 6 \"grey-zones\", where informal and illegal activities group . Saksena et al. built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core.", "built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes LCLUC . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease .", "LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts , although it is not clear if reduced species diversity necessarily increases pathogen transmission . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere.", "Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 , much of this demand is met by farms near cities , many in areas undergoing processes of peri-urbanization . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers . Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds.", "Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive backyard systems and intensive systems 15, 37 . In much of Asia there is often a mix of commercial and backyard farming at any one location . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor . Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation .", "Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. 15, 37 . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes.", "In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project.", "Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI .", "to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI . The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero.", "The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land including miscellaneous uses for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles.", "The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density 11, 14-16, 18, 19, 44, 45 . • Poultry trade and market. Towns and cities were assumed to be active trading places . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure .", "So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure . So, the distance to the nearest a national highway and b provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area . • Intermediate hosts. Duck and geese densities were calculated using total commune area . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors.", "As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus.", "We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks 47, , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk .", "We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam . Compound Topographical Index CTI, also known as Topographical Wetness Index is a measure of the tendency for water to pool. Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy.", "Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models ENM of HPAI H5N1 . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance .", "CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln A s / tan β where; A s = Area Value calculated as flow accumulation + 1 Ã pixel area in m 2 and β is the slope expressed in radians . Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health .", "Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune.", "In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose.", "Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually.", "We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level.", "Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI .", "This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI . Compound Topographical Index CTI data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation .", "Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation . Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors or their log-transform, as the case may be . Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient .", "Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient . When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term .", "Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time.", "The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 in at least one wave-place combination and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies . We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects.", "We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology . It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, .", "It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model.", "We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate lr and tree complexity tc to determine the number of trees for optimal prediction . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed.", "Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve AUC of the receiver operation characteristic ROC curve. AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates .", "AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates . We used the corrected Akaike Information Criteria AICc to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 IBM Corp., New York, 2012 for GLMM and R version 3.1.0 The R Foundation for Statistical Computing, 2014 for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations.", "For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 Tables 2-7 . The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level.", "The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 AUC = 0.69 and Wave 2 AUC = 0.77 by Gilbert et al. . Both Gilbert et al. and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta.", "and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions .", "The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk RR of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f .", "In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2.", "Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1.", "This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places.", "Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher 60-78% than at the delta levels 14-35% . In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance.", "In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable poultry outbreaks is that the data may have reporting/detection biases . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose.", "We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry.", "This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1 when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2 high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3 landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al.", "Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1.", "When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity.", "Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics e.g. . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g.", "This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g. 15, . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity .", "Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity . Mapping land-use classes at the national scale for local administrative units i.e., the 10,820 communes in Viet Nam is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited.", "Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level.", "So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors.", "CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant.", "Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere.", "The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar.", "Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes.", "Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices." ]	1,628	590	Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases EID , the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza HPAI H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances . Highly Pathogenic Avian Influenza HPAI subtype H5N1 is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility . Lederberg et al. first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants humans included of these environments, and our interactions including hygienic and therapeutic interventions with the parasites" . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems CAS . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1 peri-urban areas, as defined by Saksena et al. , 2 land-use diversity, and 3 co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time . but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes rural areas and towns, and provincial towns and cities are divided into wards urban subdistricts and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban . Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness . Other studies prioritized human population density as a satisfactory surrogate 11, , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for "village-town" . McGee identified six characteristics of desakota regions: 1 a large population of smallholder cultivators; 2 an increase in non-agricultural activities; 3 extreme fluidity and mobility of population; 4 a mixture of land uses, agriculture, cottage industries, suburban development; 5 increased participation of the female labor force; and 6 "grey-zones", where informal and illegal activities group . Saksena et al. built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes LCLUC . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts , although it is not clear if reduced species diversity necessarily increases pathogen transmission . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 , much of this demand is met by farms near cities , many in areas undergoing processes of peri-urbanization . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers . Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive backyard systems and intensive systems 15, 37 . In much of Asia there is often a mix of commercial and backyard farming at any one location . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor . Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. 15, 37 . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI . The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land including miscellaneous uses for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density 11, 14-16, 18, 19, 44, 45 . • Poultry trade and market. Towns and cities were assumed to be active trading places . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure . So, the distance to the nearest a national highway and b provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area . • Intermediate hosts. Duck and geese densities were calculated using total commune area . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks 47, , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam . Compound Topographical Index CTI, also known as Topographical Wetness Index is a measure of the tendency for water to pool. Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models ENM of HPAI H5N1 . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln A s / tan β where; A s = Area Value calculated as flow accumulation + 1 Ã pixel area in m 2 and β is the slope expressed in radians . Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI . Compound Topographical Index CTI data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation . Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors or their log-transform, as the case may be . Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient . When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 in at least one wave-place combination and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies . We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology . It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate lr and tree complexity tc to determine the number of trees for optimal prediction . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve AUC of the receiver operation characteristic ROC curve. AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates . We used the corrected Akaike Information Criteria AICc to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 IBM Corp., New York, 2012 for GLMM and R version 3.1.0 The R Foundation for Statistical Computing, 2014 for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 Tables 2-7 . The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 AUC = 0.69 and Wave 2 AUC = 0.77 by Gilbert et al. . Both Gilbert et al. and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk RR of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher 60-78% than at the delta levels 14-35% . In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable poultry outbreaks is that the data may have reporting/detection biases . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1 when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2 high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3 landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics e.g. . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g. 15, . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity . Mapping land-use classes at the national scale for local administrative units i.e., the 10,820 communes in Viet Nam is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.
What is the advantage of Boosted Regression Tree method?	The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance.	[ "Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases EID , the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza HPAI H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas.", "Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances .", "These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances . Highly Pathogenic Avian Influenza HPAI subtype H5N1 is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of \"surprise\" . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015.", "In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility . Lederberg et al. first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers.", "first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial \"adaptation and change,\" along with \"changing ecosystems\" and \"economic development and land use\" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying \"Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants humans included of these environments, and our interactions including hygienic and therapeutic interventions with the parasites\" . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the \"emerging properties\" of complex adaptive systems CAS .", "Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the \"emerging properties\" of complex adaptive systems CAS . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely.", "The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1 peri-urban areas, as defined by Saksena et al. , 2 land-use diversity, and 3 co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years .", "We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time . but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities.", "but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes rural areas and towns, and provincial towns and cities are divided into wards urban subdistricts and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term \"commune\" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work.", "For the purpose of simplicity we will henceforth use the term \"commune\" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health.", "Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban .", "Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban . Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness . Other studies prioritized human population density as a satisfactory surrogate 11, , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI.", "Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of \"ruralness\". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for \"village-town\" .", "While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of \"ruralness\". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for \"village-town\" . McGee identified six characteristics of desakota regions: 1 a large population of smallholder cultivators; 2 an increase in non-agricultural activities; 3 extreme fluidity and mobility of population; 4 a mixture of land uses, agriculture, cottage industries, suburban development; 5 increased participation of the female labor force; and 6 \"grey-zones\", where informal and illegal activities group . Saksena et al. built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core.", "built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes LCLUC . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease .", "LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts , although it is not clear if reduced species diversity necessarily increases pathogen transmission . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere.", "Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 , much of this demand is met by farms near cities , many in areas undergoing processes of peri-urbanization . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers . Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds.", "Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive backyard systems and intensive systems 15, 37 . In much of Asia there is often a mix of commercial and backyard farming at any one location . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor . Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation .", "Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. 15, 37 . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes.", "In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project.", "Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI .", "to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI . The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero.", "The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land including miscellaneous uses for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles.", "The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density 11, 14-16, 18, 19, 44, 45 . • Poultry trade and market. Towns and cities were assumed to be active trading places . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure .", "So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure . So, the distance to the nearest a national highway and b provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area . • Intermediate hosts. Duck and geese densities were calculated using total commune area . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors.", "As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus.", "We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks 47, , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk .", "We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam . Compound Topographical Index CTI, also known as Topographical Wetness Index is a measure of the tendency for water to pool. Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy.", "Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models ENM of HPAI H5N1 . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance .", "CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln A s / tan β where; A s = Area Value calculated as flow accumulation + 1 Ã pixel area in m 2 and β is the slope expressed in radians . Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health .", "Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune.", "In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose.", "Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually.", "We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level.", "Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI .", "This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI . Compound Topographical Index CTI data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation .", "Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation . Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors or their log-transform, as the case may be . Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient .", "Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient . When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term .", "Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time.", "The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 in at least one wave-place combination and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies . We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects.", "We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology . It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, .", "It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model.", "We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate lr and tree complexity tc to determine the number of trees for optimal prediction . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed.", "Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve AUC of the receiver operation characteristic ROC curve. AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates .", "AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates . We used the corrected Akaike Information Criteria AICc to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 IBM Corp., New York, 2012 for GLMM and R version 3.1.0 The R Foundation for Statistical Computing, 2014 for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations.", "For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 Tables 2-7 . The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level.", "The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 AUC = 0.69 and Wave 2 AUC = 0.77 by Gilbert et al. . Both Gilbert et al. and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta.", "and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions .", "The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk RR of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f .", "In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2.", "Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1.", "This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places.", "Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher 60-78% than at the delta levels 14-35% . In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance.", "In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable poultry outbreaks is that the data may have reporting/detection biases . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose.", "We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry.", "This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1 when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2 high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3 landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al.", "Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1.", "When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity.", "Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics e.g. . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g.", "This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g. 15, . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity .", "Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity . Mapping land-use classes at the national scale for local administrative units i.e., the 10,820 communes in Viet Nam is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited.", "Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level.", "So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors.", "CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant.", "Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere.", "The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar.", "Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes.", "Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices." ]	1,628	591	Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases EID , the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza HPAI H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances . Highly Pathogenic Avian Influenza HPAI subtype H5N1 is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility . Lederberg et al. first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants humans included of these environments, and our interactions including hygienic and therapeutic interventions with the parasites" . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems CAS . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1 peri-urban areas, as defined by Saksena et al. , 2 land-use diversity, and 3 co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time . but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes rural areas and towns, and provincial towns and cities are divided into wards urban subdistricts and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban . Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness . Other studies prioritized human population density as a satisfactory surrogate 11, , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for "village-town" . McGee identified six characteristics of desakota regions: 1 a large population of smallholder cultivators; 2 an increase in non-agricultural activities; 3 extreme fluidity and mobility of population; 4 a mixture of land uses, agriculture, cottage industries, suburban development; 5 increased participation of the female labor force; and 6 "grey-zones", where informal and illegal activities group . Saksena et al. built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes LCLUC . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts , although it is not clear if reduced species diversity necessarily increases pathogen transmission . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 , much of this demand is met by farms near cities , many in areas undergoing processes of peri-urbanization . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers . Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive backyard systems and intensive systems 15, 37 . In much of Asia there is often a mix of commercial and backyard farming at any one location . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor . Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. 15, 37 . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI . The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land including miscellaneous uses for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density 11, 14-16, 18, 19, 44, 45 . • Poultry trade and market. Towns and cities were assumed to be active trading places . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure . So, the distance to the nearest a national highway and b provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area . • Intermediate hosts. Duck and geese densities were calculated using total commune area . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks 47, , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam . Compound Topographical Index CTI, also known as Topographical Wetness Index is a measure of the tendency for water to pool. Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models ENM of HPAI H5N1 . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln A s / tan β where; A s = Area Value calculated as flow accumulation + 1 Ã pixel area in m 2 and β is the slope expressed in radians . Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI . Compound Topographical Index CTI data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation . Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors or their log-transform, as the case may be . Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient . When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 in at least one wave-place combination and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies . We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology . It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate lr and tree complexity tc to determine the number of trees for optimal prediction . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve AUC of the receiver operation characteristic ROC curve. AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates . We used the corrected Akaike Information Criteria AICc to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 IBM Corp., New York, 2012 for GLMM and R version 3.1.0 The R Foundation for Statistical Computing, 2014 for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 Tables 2-7 . The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 AUC = 0.69 and Wave 2 AUC = 0.77 by Gilbert et al. . Both Gilbert et al. and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk RR of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher 60-78% than at the delta levels 14-35% . In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable poultry outbreaks is that the data may have reporting/detection biases . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1 when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2 high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3 landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics e.g. . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g. 15, . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity . Mapping land-use classes at the national scale for local administrative units i.e., the 10,820 communes in Viet Nam is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.
What is the relationship between land use and emergence of HPAI H5N1?	high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks	[ "Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases EID , the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza HPAI H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas.", "Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances .", "These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances . Highly Pathogenic Avian Influenza HPAI subtype H5N1 is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of \"surprise\" . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015.", "In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility . Lederberg et al. first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers.", "first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial \"adaptation and change,\" along with \"changing ecosystems\" and \"economic development and land use\" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying \"Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants humans included of these environments, and our interactions including hygienic and therapeutic interventions with the parasites\" . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the \"emerging properties\" of complex adaptive systems CAS .", "Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the \"emerging properties\" of complex adaptive systems CAS . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely.", "The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1 peri-urban areas, as defined by Saksena et al. , 2 land-use diversity, and 3 co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years .", "We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time . but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities.", "but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes rural areas and towns, and provincial towns and cities are divided into wards urban subdistricts and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term \"commune\" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work.", "For the purpose of simplicity we will henceforth use the term \"commune\" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health.", "Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban .", "Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban . Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness . Other studies prioritized human population density as a satisfactory surrogate 11, , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI.", "Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of \"ruralness\". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for \"village-town\" .", "While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of \"ruralness\". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for \"village-town\" . McGee identified six characteristics of desakota regions: 1 a large population of smallholder cultivators; 2 an increase in non-agricultural activities; 3 extreme fluidity and mobility of population; 4 a mixture of land uses, agriculture, cottage industries, suburban development; 5 increased participation of the female labor force; and 6 \"grey-zones\", where informal and illegal activities group . Saksena et al. built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core.", "built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes LCLUC . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease .", "LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts , although it is not clear if reduced species diversity necessarily increases pathogen transmission . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere.", "Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 , much of this demand is met by farms near cities , many in areas undergoing processes of peri-urbanization . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers . Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds.", "Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive backyard systems and intensive systems 15, 37 . In much of Asia there is often a mix of commercial and backyard farming at any one location . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor . Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation .", "Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. 15, 37 . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes.", "In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project.", "Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI .", "to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI . The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero.", "The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land including miscellaneous uses for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles.", "The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density 11, 14-16, 18, 19, 44, 45 . • Poultry trade and market. Towns and cities were assumed to be active trading places . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure .", "So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure . So, the distance to the nearest a national highway and b provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area . • Intermediate hosts. Duck and geese densities were calculated using total commune area . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors.", "As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus.", "We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks 47, , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk .", "We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam . Compound Topographical Index CTI, also known as Topographical Wetness Index is a measure of the tendency for water to pool. Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy.", "Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models ENM of HPAI H5N1 . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance .", "CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln A s / tan β where; A s = Area Value calculated as flow accumulation + 1 Ã pixel area in m 2 and β is the slope expressed in radians . Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health .", "Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune.", "In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose.", "Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually.", "We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level.", "Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI .", "This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI . Compound Topographical Index CTI data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation .", "Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation . Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors or their log-transform, as the case may be . Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient .", "Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient . When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term .", "Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time.", "The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 in at least one wave-place combination and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies . We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects.", "We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology . It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, .", "It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model.", "We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate lr and tree complexity tc to determine the number of trees for optimal prediction . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed.", "Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve AUC of the receiver operation characteristic ROC curve. AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates .", "AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates . We used the corrected Akaike Information Criteria AICc to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 IBM Corp., New York, 2012 for GLMM and R version 3.1.0 The R Foundation for Statistical Computing, 2014 for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations.", "For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 Tables 2-7 . The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level.", "The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 AUC = 0.69 and Wave 2 AUC = 0.77 by Gilbert et al. . Both Gilbert et al. and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta.", "and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions .", "The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk RR of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f .", "In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2.", "Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1.", "This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places.", "Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher 60-78% than at the delta levels 14-35% . In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance.", "In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable poultry outbreaks is that the data may have reporting/detection biases . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose.", "We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry.", "This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1 when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2 high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3 landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al.", "Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1.", "When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity.", "Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics e.g. . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g.", "This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g. 15, . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity .", "Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity . Mapping land-use classes at the national scale for local administrative units i.e., the 10,820 communes in Viet Nam is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited.", "Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level.", "So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors.", "CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant.", "Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere.", "The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar.", "Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes.", "Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices." ]	1,628	592	Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases EID , the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza HPAI H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances . Highly Pathogenic Avian Influenza HPAI subtype H5N1 is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility . Lederberg et al. first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants humans included of these environments, and our interactions including hygienic and therapeutic interventions with the parasites" . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems CAS . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1 peri-urban areas, as defined by Saksena et al. , 2 land-use diversity, and 3 co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time . but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes rural areas and towns, and provincial towns and cities are divided into wards urban subdistricts and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban . Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness . Other studies prioritized human population density as a satisfactory surrogate 11, , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for "village-town" . McGee identified six characteristics of desakota regions: 1 a large population of smallholder cultivators; 2 an increase in non-agricultural activities; 3 extreme fluidity and mobility of population; 4 a mixture of land uses, agriculture, cottage industries, suburban development; 5 increased participation of the female labor force; and 6 "grey-zones", where informal and illegal activities group . Saksena et al. built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes LCLUC . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts , although it is not clear if reduced species diversity necessarily increases pathogen transmission . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 , much of this demand is met by farms near cities , many in areas undergoing processes of peri-urbanization . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers . Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive backyard systems and intensive systems 15, 37 . In much of Asia there is often a mix of commercial and backyard farming at any one location . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor . Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. 15, 37 . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI . The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land including miscellaneous uses for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density 11, 14-16, 18, 19, 44, 45 . • Poultry trade and market. Towns and cities were assumed to be active trading places . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure . So, the distance to the nearest a national highway and b provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area . • Intermediate hosts. Duck and geese densities were calculated using total commune area . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks 47, , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam . Compound Topographical Index CTI, also known as Topographical Wetness Index is a measure of the tendency for water to pool. Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models ENM of HPAI H5N1 . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln A s / tan β where; A s = Area Value calculated as flow accumulation + 1 Ã pixel area in m 2 and β is the slope expressed in radians . Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI . Compound Topographical Index CTI data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation . Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors or their log-transform, as the case may be . Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient . When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 in at least one wave-place combination and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies . We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology . It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate lr and tree complexity tc to determine the number of trees for optimal prediction . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve AUC of the receiver operation characteristic ROC curve. AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates . We used the corrected Akaike Information Criteria AICc to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 IBM Corp., New York, 2012 for GLMM and R version 3.1.0 The R Foundation for Statistical Computing, 2014 for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 Tables 2-7 . The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 AUC = 0.69 and Wave 2 AUC = 0.77 by Gilbert et al. . Both Gilbert et al. and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk RR of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher 60-78% than at the delta levels 14-35% . In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable poultry outbreaks is that the data may have reporting/detection biases . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1 when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2 high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3 landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics e.g. . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g. 15, . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity . Mapping land-use classes at the national scale for local administrative units i.e., the 10,820 communes in Viet Nam is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.
Where is the highest risk of HPAI H5N1 like disease emergence?	Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely.	[ "Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases EID , the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza HPAI H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas.", "Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances .", "These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances . Highly Pathogenic Avian Influenza HPAI subtype H5N1 is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of \"surprise\" . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015.", "In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility . Lederberg et al. first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers.", "first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial \"adaptation and change,\" along with \"changing ecosystems\" and \"economic development and land use\" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying \"Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants humans included of these environments, and our interactions including hygienic and therapeutic interventions with the parasites\" . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the \"emerging properties\" of complex adaptive systems CAS .", "Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the \"emerging properties\" of complex adaptive systems CAS . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely.", "The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1 peri-urban areas, as defined by Saksena et al. , 2 land-use diversity, and 3 co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years .", "We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time . but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities.", "but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes rural areas and towns, and provincial towns and cities are divided into wards urban subdistricts and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term \"commune\" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work.", "For the purpose of simplicity we will henceforth use the term \"commune\" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health.", "Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban .", "Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban . Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness . Other studies prioritized human population density as a satisfactory surrogate 11, , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI.", "Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of \"ruralness\". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for \"village-town\" .", "While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of \"ruralness\". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for \"village-town\" . McGee identified six characteristics of desakota regions: 1 a large population of smallholder cultivators; 2 an increase in non-agricultural activities; 3 extreme fluidity and mobility of population; 4 a mixture of land uses, agriculture, cottage industries, suburban development; 5 increased participation of the female labor force; and 6 \"grey-zones\", where informal and illegal activities group . Saksena et al. built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core.", "built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes LCLUC . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease .", "LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts , although it is not clear if reduced species diversity necessarily increases pathogen transmission . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere.", "Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 , much of this demand is met by farms near cities , many in areas undergoing processes of peri-urbanization . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers . Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds.", "Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive backyard systems and intensive systems 15, 37 . In much of Asia there is often a mix of commercial and backyard farming at any one location . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor . Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation .", "Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. 15, 37 . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes.", "In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project.", "Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI .", "to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI . The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero.", "The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land including miscellaneous uses for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles.", "The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density 11, 14-16, 18, 19, 44, 45 . • Poultry trade and market. Towns and cities were assumed to be active trading places . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure .", "So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure . So, the distance to the nearest a national highway and b provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area . • Intermediate hosts. Duck and geese densities were calculated using total commune area . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors.", "As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus.", "We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks 47, , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk .", "We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam . Compound Topographical Index CTI, also known as Topographical Wetness Index is a measure of the tendency for water to pool. Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy.", "Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models ENM of HPAI H5N1 . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance .", "CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln A s / tan β where; A s = Area Value calculated as flow accumulation + 1 Ã pixel area in m 2 and β is the slope expressed in radians . Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health .", "Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune.", "In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose.", "Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually.", "We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level.", "Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI .", "This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI . Compound Topographical Index CTI data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation .", "Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation . Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors or their log-transform, as the case may be . Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient .", "Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient . When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term .", "Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time.", "The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 in at least one wave-place combination and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies . We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects.", "We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology . It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, .", "It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model.", "We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate lr and tree complexity tc to determine the number of trees for optimal prediction . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed.", "Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve AUC of the receiver operation characteristic ROC curve. AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates .", "AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates . We used the corrected Akaike Information Criteria AICc to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 IBM Corp., New York, 2012 for GLMM and R version 3.1.0 The R Foundation for Statistical Computing, 2014 for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations.", "For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 Tables 2-7 . The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level.", "The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 AUC = 0.69 and Wave 2 AUC = 0.77 by Gilbert et al. . Both Gilbert et al. and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta.", "and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions .", "The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk RR of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f .", "In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2.", "Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1.", "This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places.", "Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher 60-78% than at the delta levels 14-35% . In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance.", "In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable poultry outbreaks is that the data may have reporting/detection biases . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose.", "We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry.", "This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1 when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2 high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3 landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al.", "Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1.", "When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity.", "Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics e.g. . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g.", "This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g. 15, . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity .", "Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity . Mapping land-use classes at the national scale for local administrative units i.e., the 10,820 communes in Viet Nam is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited.", "Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level.", "So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors.", "CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant.", "Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere.", "The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar.", "Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes.", "Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices." ]	1,628	589	Building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases EID , the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. The model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. We developed and tested a model of the emergence of highly pathogenic avian influenza HPAI H5N1 based on suspected convergence factors that are mainly associated with land-use change. Building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. Our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher HPAI H5N1 emergence risk. The work highlights that peri-urban areas of Viet Nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. We also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. Similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. These results support the convergence hypothesis in general and demonstrate the potential to improve EID prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. Text: Two decades after the Institute of Medicine's seminal report recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances . Highly Pathogenic Avian Influenza HPAI subtype H5N1 is the most significant newly emerging pandemic disease since HIV/AIDS. Its eruption in Southeast Asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" . In this same year that IOM had published its final report on microbial threats which highlighted H5N1's successful containment in Hong Kong in 1997 , massive outbreaks occurred in Southeast Asia where it remains endemic, along with Egypt's Nile Delta. Since 2003, HPAI H5N1 has killed millions of poultry in countries throughout Asia, Europe, and Africa, and 402 humans have died from it in sixteen countries according to WHO data as of January 2015. The threat of a pandemic resulting in millions of human cases worldwide remains a possibility . Lederberg et al. first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' . The model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. Microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. Joshua Lederberg, the major intellectual force behind the studies summed-up saying "Ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants humans included of these environments, and our interactions including hygienic and therapeutic interventions with the parasites" . Combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. One approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems CAS . The convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. These associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. The initial HPAI H5N1 outbreaks in Vietnam represent an ideal opportunity to adapt and test a CAS-convergence model. Emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. Specifically we hypothesized a positive association between the presence of HPAI outbreaks in poultry at the commune level and: 1 peri-urban areas, as defined by Saksena et al. , 2 land-use diversity, and 3 co-location of intensive and extensive systems of poultry. We used the presence or absence at the commune level of HPAI H5N1 outbreaks in poultry as the dependent variable. Vietnam experienced its first HPAI H5N1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. We used data from the Viet Nam 2006 Agricultural Census to develop an urbanicity classification that used data collected at a single point in time . but across space 10,820 communes to infer processes of change urbanization, land-use diversification, and poultry intensification . The 58 provinces in Vietnam not counting the 5 urban provinces that are governed centrally are divided into rural districts, provincial towns, and provincial cities. Rural districts are further divided into communes rural areas and towns, and provincial towns and cities are divided into wards urban subdistricts and communes. A commune in Viet Nam is thus the third level administrative subdivision, consisting of villages/hamlets. For the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. We included risk factors documented in previous work. We also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. For this purpose we chose to study the Red River and Mekong River deltas, well known hot spots of the disease. Hence we conducted two sets of analyses waves 1 and 2 for three places nation, Red River Delta, and Mekong Delta producing a total of 6 wave-place analyses. Data on outbreaks were obtained from the publicly available database of Viet Nam's Department of Animal Health. Given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. We used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon . Very few empirical studies have attempted to determine whether urbanization is related to EID outbreaks or whether urbanization is associated primarily with other factors related to EID outbreaks. One immediate problem researchers face is defining what is rural, urban, and transitional i.e., peri-urban . Some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness . Other studies prioritized human population density as a satisfactory surrogate 11, , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. Spencer examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. He found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of HPAI. These studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. Still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' . While these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". Perhaps the best known model of peri-urbanization is McGee's concept of desakota Indonesian for "village-town" . McGee identified six characteristics of desakota regions: 1 a large population of smallholder cultivators; 2 an increase in non-agricultural activities; 3 extreme fluidity and mobility of population; 4 a mixture of land uses, agriculture, cottage industries, suburban development; 5 increased participation of the female labor force; and 6 "grey-zones", where informal and illegal activities group . Saksena et al. built on McGee's desakota concepts and data from the 2006 Viet Nam Agricultural Census to establish an urbanicity classification. That study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. This project used the Saksena classification to assess associations between urbanicity classes, other risks factors, and HPAI outbreaks. Researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes LCLUC . LCLUC such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes number of land covers or land uses per unit of land . The importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease . Landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species Furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts , although it is not clear if reduced species diversity necessarily increases pathogen transmission . Rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with HPAI H5N1 outbreaks. Human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. As theorized by von Thünen in 1826 , much of this demand is met by farms near cities , many in areas undergoing processes of peri-urbanization . Due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in Southeast Asia and compete with existing small backyard farmers . Large, enterprise-scale 15,000-100,000 birds operations are still rare in Viet Nam only 33 communes have such a facility . On the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. Recent studies have examined the relative role of extensive backyard systems and intensive systems 15, 37 . In much of Asia there is often a mix of commercial and backyard farming at any one location . Experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor . Intensive systems allow for virus evolution e.g. Low Pathogenic Avian Influenza to HPAI and transformation, while extensive systems allow for environmental persistence and circulation . Previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. 15, 37 . In isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. Intensive and extensive systems in Viet Nam have their own fairly well defined flock sizes. A diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of HPAI H5N1 outbreaks at the commune level. This study investigated for the 10,820 communes of Viet Nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the HPAI virus. Many of these variables were identified based on earlier studies of HPAI as reviewed in Gilbert and Pfeiffer . Three novel variables were included based on hypotheses generated by this project. All variables were measured or aggregated to the commune level. The novel variables were: • Degree of urbanization: We used the urbanicity classification developed by Saksena et al. to define the urban character of each commune. The classification framework is based on four characteristics: 1 percentage of households whose main income is from agriculture, aquaculture and forestry, 2 percentage of households with modern forms of toilets, 3 percentage of land under agriculture, aquaculture and forestry and 4 the Normalized Differentiated Vegetation Index NDVI . The three-way classification enabled testing for non-linear and non-monotonous responses. • Land-use diversity: We measured land-use diversity using the Gini-Simpson Diversity Index . The Gini-Simpson Diversity Index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. In situations with only one class complete homogeneity the Gini-Simpson index would have a value equal to zero. Such diversity indices have been used to measure land-use diversity . We used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land including miscellaneous uses for which data were collected in the 2006 Agricultural Census. The area under the last class was calculated as the difference between the total area and the sum of the first four classes. The following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • Human population related transmission. Human population density 11, 14-16, 18, 19, 44, 45 . • Poultry trade and market. Towns and cities were assumed to be active trading places . So, the distance to the nearest town/city was used as indicator of poultry trade. Trade is facilitated by access to transportation infrastructure . So, the distance to the nearest a national highway and b provincial highway was used as indicator of transportation infrastructure. • Disease introduction and amplification. The densities of chicken were calculated based on commune area . • Intermediate hosts. Duck and geese densities were calculated using total commune area . As previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • Agro-ecological and environmental risk factors. Previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers . We used percentage of land under rice cultivation as a measure of extent. Rice cropping intensity is also a known risk factor . We used the mean number of rice crops per year as a measure of intensity. The extent of aquaculture is a known risk factor , possibly because water bodies offer routes for transmission and persistence of the virus. The percentage of land under aquaculture was used as a metric. Proximity to water bodies increases the risk of outbreaks 47, , possibly by increasing the chance of contact between wild water birds and domestic poultry. We measured the distance between the commune and the nearest: a lake and b river. Climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk . Elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in Vietnam . Compound Topographical Index CTI, also known as Topographical Wetness Index is a measure of the tendency for water to pool. Studies in Thailand and elsewhere have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. In the absence of reliable and inexpensive data on the extent of surface water we used CTI as a proxy. CTI has been used in Ecological Niche Models ENM of HPAI H5N1 . However, given the nature of ENM studies, the effect of CTI as a risk factor has been unknown so far. CTI has been used as a risk factor in the study of other infectious and non-infectious diseases . Some studies have shown that at local scales, the slope of the terrain a component of CTI was significantly correlated with reservoir species dominance . CTI is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. CTI is computed as follows: CTI = ln A s / tan β where; A s = Area Value calculated as flow accumulation + 1 Ã pixel area in m 2 and β is the slope expressed in radians . Though previous studies have indicated that Normalized Difference Vegetation Index NDVI is a risk factor , we did not include it explicitly in our models, as the urban classification index we used included NDVI . We obtained commune level data on HPAI H5N1 outbreaks from the publicly available database of the Department of Animal Health . Viet Nam experienced its first major epidemic waves between December 2003 and February 2006 . We chose to study the first wave Wave 1 that ended in February 2004 and the second wave Wave 2 that occurred between December 2004 and April 2005. In Wave 1, 21% of the communes and in Wave 2, 6% of the communes experienced outbreaks. We used data from the 1999 Population Census of Viet Nam to estimate human population per commune. We relied on data from two Agriculture Censuses of Viet Nam. This survey is conducted every five years covering all rural households and those peri-urban households that own farms. Thus about three-fourths of all of the country's households are included. The contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. Commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. Detailed economic data are collected for large farms. We used the 2006 Agriculture Census for most variables because the first three epidemic waves occurred between the Agricultural Censuses of 2001 and 2006 but were closer in time to the 2006 census . However, for data on poultry numbers we used the 2001 Agriculture Census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. However, in 2004, after the first wave of the H5N1 epidemic, the poultry population fell 15%. Only by mid-2008 did the poultry population return close to pre-epidemic levels. Thus, we considered the poultry population data from the 2001 census to be more representative. We aggregated census household data to the commune level. A three-way classification of the rural-to-urban transition was based on a related study . Raster data on annual mean temperature and precipitation were obtained from the World-Clim database and converted to commune level data. The bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution . This public database provides data on the average climatic conditions of the period 1950-2000. Elevation was generated from SRTM 90 meter Digital Elevation Models DEM acquired from the Consortium for Spatial Information CGIAR-CSI . Compound Topographical Index CTI data were generated using the Geomorphometry and Gradient Metrics Toolbox for Arc-GIS 10.1. Prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. Illogical values occurred mainly less than 1% of the cases for land-related variables such as percentage of commune land under a particular type of land use. Next we tested each variable for normality using the BestFit software Palisade Corporation . Most of the variables were found to follow a log-normal distribution and a log-transform was used on them. We then examined the bi-variate correlations between all the risk factors or their log-transform, as the case may be . Correlations were analyzed separately for each place. Certain risk factors were then eliminated from consideration when \|r\| ! 0.5 r is the Pearson correlation coefficient . When two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. Notably, we excluded a elevation correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index , b human population density correlated with elevation and CTI , c chicken density only at national level, correlated with CTI , d duck and goose density correlated with elevation, chicken density, percentage land under paddy, land use diversity index and CTI , e annual temperature correlated with elevation and CTI and f cropping intensity correlated with percentage land under paddy . Considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1 multi-level Generalized Linear Mixed Model GLMM and 2 Boosted Regression trees BRT with an autoregressive term . GLMM is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while BRT is a 'space' oriented approach that accounts for the effects of physical proximity. We began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the Euclidean distance . The limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ h . To determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. We finally included only those variables whose coefficient had a significance value p 0.2 in at least one wave-place combination and we noted the sign of the coefficient. This choice of p value for screening risk factors is common in similar studies . We used a two-level GLMM communes nested under districts to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. We used robust standard errors for tests of fixed effects. Boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of HPAI H5N1 occurrence and determine the relative influence of each risk factor to the HPAI H5N1 occurrence. This method was developed recently and applied widely for distribution prediction in various fields of ecology . It is widely used for species distribution modeling where only the sites of occurrence of the species are known . The method has been applied in numerous studies for predicting the distribution of HPAI H5N1 disease 16, 51, . BRT utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model . The advantage of BRT is that it applies stochastic processes that include probabilistic components to improve predictive performance. We used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. The sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. Two important parameters specified in the BRT model are learning rate lr and tree complexity tc to determine the number of trees for optimal prediction . In our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. Other advantages of BRT include its insensitivity to co-linearity and non-linear responses. However, for the sake of consistency with the GLMM method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. In the GLMM models we used p 0.05 to identify significant risk factors. The predictive performances of the models were assessed by the area under the curve AUC of the receiver operation characteristic ROC curve. AUC is a measure of the overall fit of the model that varies from 0.5 chance event to 1.0 perfect fit . A comparison of AUC with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates . We used the corrected Akaike Information Criteria AICc to compare each GLMM model with and without its respective suite of fixed predictors. We used SPSS version 21 IBM Corp., New York, 2012 for GLMM and R version 3.1.0 The R Foundation for Statistical Computing, 2014 for the BRT. For calculating the spatial correlogram we used the spdep package of R. The fourteen predictor variables we modeled see tables were all found to be significantly associated with HPAI H5N1 outbreaks p 0.2 in at least one wave-place combination based on univariate analysis but including the autoregressive term Table 1 . Land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the GLMM models, as measured by the AUC, is very good with AUC values ranging from 0.802 to 0.952 Tables 2-7 . The predictive power of the national models was higher than that of the delta models. The predictive power of the BRT models is good, with AUCs ranging from 0.737 to 0.914. The BRT models also had a better predictive power at the national level than at the delta level. These values are higher than those reported for Wave 1 AUC = 0.69 and Wave 2 AUC = 0.77 by Gilbert et al. . Both Gilbert et al. and this study found that at the national level the predictive performance for Wave 2 was higher than that for Wave 1. Wave 2 mainly affected the Mekong River Delta. Previous studies indicated the duck density was an important predictor ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. Both the GLMM and BRT models found annual precipitation to be a significant factor. The GLMM model indicated a negative association; similar to what was found by studies in China and in the Red River Delta . A global study of human cases also found occurrence to be higher under drier conditions . Generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. The unadjusted Relative Risk RR of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for Waves 1 and 2, respectively. In terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the Compound Topographical Index CTI to be highest in peri-urban areas Fig 1a-1e . We also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower Fig 1f . The urbanicity variable alone, however, was not found to be significantly associated with HPAI H5N1 in any place according to the GLMM model except for the urban level in Red River Delta for Wave 2 and in the Mekong River Delta for Wave 1. The BRT model ranked urbanicity as one of the least influential variables. Land-use diversity was found to be significantly associated with HPAI H5N1 in both waves for Viet Nam according to the GLMM model, but at the delta level the association was significant only for Wave 2 in the Mekong River Delta. The BRT model indicated that land-use diversity highly influenced HPAI H5N1 at the national level in Wave 2. For the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. Both the GLMM and BRT models indicated that the diversity of chicken flock-size had a strong association with HPAI H5N1 for both waves at the national level. This was generally found to be true at the delta levels with some exceptions. The diversity of duck and goose flock size was also significantly associated with HPAI H5N1 in all places, but the associations were much stronger in Wave 2 than in Wave 1. The GLMM model indicated that the CTI had a very strong association with HPAI H5N1 at the national level in both waves although this was not true in the two deltas. The CTI is a steady state wetness index commonly used to quantify topographic control on hydrological processes. Accumulation numbers in flat areas, like deltas, are very large; hence the CTI was not a relevant variable in the GLMM model in these areas. The BRT model however indicated that CTI had middle to low influence in all waves and places. We found very high spatial clustering effects as indicated by the fact that in all waves and places the BRT model found the spatial autocorrelation term to have the highest rank of influence. As expected, the relative influence of the autocorrelation term at the national level was higher 60-78% than at the delta levels 14-35% . In the GLMM models we found the Akaike Information Criterion AIC using the entire set of 14 variables to be much lower than the AICs of a GLMM model without fixed effects. This indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. A limitation of using surveillance methods for the dependent variable poultry outbreaks is that the data may have reporting/detection biases . Under-reporting/detection in rural areas as compared to peri-urban areas is possible. We believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. Previous studies have tended to use human population density as a proxy for this purpose. In our study we found a strong association between human population density and urbanicity. But we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. This study explored the validity of a general model for disease emergence that combined the IOM 'convergence model' and the social-ecological systems model , for investigating the specific case of HPAI in Vietnam. We sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. Our results generally support the hypothesis that social-ecological system transformations are associated with H5NI outbreaks in poultry. The results presented here highlight three main findings: 1 when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2 high land-use diversity landscapes, a variable not previously considered in spatial studies of HPAI H5N1, are at significantly greater risk for HPAI H5N1 outbreaks; as are 3 landscapes where intensive and extensive forms of poultry production are co-located. Only one other study has explicitly examined urbanicity in the context of HPAI H5N1. Loth et al. found peri-urban areas in Indonesia were significantly associated with HPAI H5N1 cases, even based on multivariate models. Our study, however, attempted both to associate HPAI H5N1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. When those features i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture are included in multivariate models, the role of the urbanization variable per se diminishes. We found in the main river deltas in Viet Nam Red River and Mekong , urbanization had no significant association with HPAI H5N1. This may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. This is the first study to examine land-use diversity as a risk factor for HPAI H5N1. Measured by the Gini-Simpson Diversity Index of the five land-use classes on which data were collected in the 2006 Viet Nam Agricultural Census, and the presence or absence of HPAI outbreaks at the commune level, our results indicate a strong association between land-use diversity and HPAI H5N1 at the national level and in the Mekong River Delta. This metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. Our results are similar to what has been observed by studies of other EIDs using fragmentation metrics e.g. . This is one of the few studies, however, to link landscape fragmentation to an EID disease in poultry and not just to the vector and/or hosts of the EID. Previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization e.g. 15, . This study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. Future studies need to examine the biological causal mechanisms in this context. We suggest that national census data particularly agricultural censuses compiled at local levels of administration provide valuable information that are not available from remotely sensed data such as poultry densities or require a large amount of labor to map at national to larger scales land-use diversity . Mapping land-use classes at the national scale for local administrative units i.e., the 10,820 communes in Viet Nam is not an insignificant task. Future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape . Vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. While other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. Ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. Another important contribution of this study was the discovery of the importance of CTI. So far CTI had been used only in ecological niche modeling studies of HPAI H5N1; the specific role and direction of influence of CTI had has so far been unknown. Our study, the first to use CTI as a risk factor, found it had a large positive influence on HPAI H5N1 risk at the national level. Previous studies have highlighted the role of surface water extent in the persistence and transmission of the HPAI H5N1 virus. These studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. CTI on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a GIS database. The national and regional delta models differed quite considerably, both in terms of performance and significant risk factors. In the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. This suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the Mekong Delta . Though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled NDVI median May-October, buffalo density and sweet potato yield. Another study in the Red River Delta found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. We speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. Improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. The differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. Our study has the potential to inform the design of future research related to the epidemiology of other EIDs in Viet Nam and elsewhere. For example, we speculate that in Southeast Asia, Japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation , may also show a strong association with peri-urbanization. In some areas of Asia these ecological conditions occur near, or occasionally within, urban centers. Likewise, Hantaan virus, the cause of Korean hemorrhagic fever, is associated with the field mouse Apodemus agrarius and rice harvesting in fields where the rodents are present . Our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. Hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, CTI, and distance to infrastructure. Our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as Newcastle disease. Finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates IOM's convergence model with the subsequently proposed social-ecological systems and EID framework. Thus, convergence represents the coalescence in time and space of processes associated with land-cover and land-use changes. Project results question whether the urban/rural land-use dichotomy is useful when large areas and parts of the population are caught between the two. Planners need better tools for mapping the rural-urban transition, and for understanding how the specific nature of peri-urban environments creates elevated health risk that require adaptation of existing planning, land use, and development practices.

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